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Mass screening test for mucopolysaccharidoses using the 1,9-dimethylmethylene blue method:
Clinica Chimica Acta 264 (1997) 245–250
Short communication
Mass screening test for mucopolysaccharidoses using the 1,9-dimethylmethylene blue method: Positive interference from paper diapers a, a a a Shoko Iwata *, Kazuko Sukegawa , Toshiya Sasaki , Mie Kokuryu , Sumiko Yamasita b , Akio Noma b , Shinichi Iwasa c , Naomi Kondo a , Tadao Orii a , d a
Department of Pediatrics, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500, Japan b Clinical Laboratory, Gifu University Hospital, 40 Tsukasa-machi, Gifu 500, Japan c Iwasa Hospital, 161 -1 Nagara Fukumitsu, Gifu 502, Japan d Chubu Gakuin University, 4909 -3 Kurachi, Seki, Gifu 501 -32, Japan Received 25 July 1996; received in revised form 1 April 1997; accepted 15 April 1997
Keywords: Mucopolysaccharidosis; 1,9-Dimethylmethylene blue; Mass screening; Glycosaminoglycans
1. Introduction The mucopolysaccharidoses (MPS) are a group of disorders caused by a deficiency in the lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs). The undegraded GAGs are stored in lysosomes and subsequently are excreted in the urine. Quantitative measurement of urinary GAGs is therefore available for diagnosis of the MPS. The clinical manifestations of the MPS are coarse facies, skeletal deformities, hepatosplenomegaly, joint stiffness and variable degrees of psychomotor developmental delay [1]. Bone marrow transplantation (BMT) has been proposed as a treatment for MPS, both to correct the bone marrow-derived cells in the recipient by
*Corresponding author. Tel.: 81-58-265-1241 ext. 2819; fax: 81-58-265-9011. 0009-8981 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved. PII S0009-8981( 97 )00084-3
246
S. Iwata et al. / Clinica Chimica Acta 264 (1997) 245 – 250
replacement, and also to correct other cells which take up enzyme secreted by the bone marrow-derived cells, especially macrophages [2]. Mass screening for MPS in early infancy could provide a method for presymptomatic diagnosis, and it could realize early BMT, in which clinical outcome would be better than in BMT after clinical manifestation became apparent [3–5]. We have proposed organizing mass screening for MPS in Japan and a pilot study is in progress in the Gifu Prefecture using the dimethylmethylene blue (DMB) method. In this study, we have observed new positive interference from paper diapers. We describe our findings in the pilot study of mass screening for MPS.
2. Materials and methods
2.1. Collection of urine specimens Urine samples were collected from 44 060 infants at 6 months of age. A plastic sheet was placed on the diaper and a piece of cotton was put on it. Such a diaper was attached to the infant and, after the cotton was dampened with urine, it was removed. The urine sample was squeezed from the cotton and was placed in a plastic bottle. It was returned by mail and stored at 2 208C until assay. Informed consent was obtained from the parents of the infants studied.
2.2. Pilot study for mucopolysaccharidoses in the Gifu Prefecture Over a three-year period from 1993–1995, urine samples from 6-month-old infants were screened. Initial screening was carried out using the DMB method. At first, the upper cut-off point was selected arbitrarily and was set at 400 mg of GAG / g of creatinine. The current limit was selected in the light of experience gained from the initial part of the prospective trial. When the value of urinary GAGs was over the cut-off point, a second sample was re-tested by the same method. After the second test, a third assay was carried out with the same samples using the cetylpyridinium chloride (CPC) / carbazol method and monodimensional electrophoresis [6,7].
2.3. Influence of paper diapers To study the influence of a paper diaper on the DMB reaction, the products from six manufacturers were analyzed. A 400-ml volume of urine was absorbed on the paper diaper, incubated at 378C for 12 h, squeezed and assayed by the DMB method. As a control sample, urine was incubated at 378C for 12 h and
S. Iwata et al. / Clinica Chimica Acta 264 (1997) 245 – 250
247
assayed by the DMB method. Since crosslinked acrylic acid polymer sodium salt (acrylic acid polymer) is used as the absorbent of the paper diaper, we analyzed acrylic acid polymer as a candidate of positive interference on the DMB method. A 1-g amount of crosslinked acrylic acid polymer sodium salt (SANYOKASEI, Kyoto, Japan) was dissolved in 200 ml of physiological saline, incubated at 378C for 12 h, filtered and the eluate was assayed by the DMB method. Tissue paper and acrylic acid were used as references.
2.4. Measurement of urinary GAG The DMB and cetylpyridinium chloride (CPC) / carbazole-borate assays were carried out using a modification of the method of de Jong et al. [8] and of Bitter and Muir [6], respectively. Urinary creatinine was measured by the Jaffe method [9]. The measurement was automatically performed with a MR 5000 plate reader (Dynatech, USA).
3. Results
3.1. Mass screening pilot study for MPS We tested 44 060 infants at 6 months of age. With an upper cut-off limit of 400 mg of GAG / g of creatinine, 842 infants (1.9%) were positive. A second sample was requested for the positive individuals, of which 671 were re-tested: In the second screening, 27 cases were found to be positive and in the third screening using the CPC / carbazole method, all specimens were found to be negative (Table 1). To determine the range of GAG excretion in this population, we analyzed 10 000 samples obtained from presumed-normal 6-month-old infants using the DMB method. By a non-parametric method, the 95% confidence interval of this population was 71.0–465.5 mg of GAG / g of creatinine (Fig. 1).
Table 1 Pilot mass screening study for MPS in the Gifu Prefecture
First screening Second screening Third screening
n
Positive (%)
44 060 671 27
842 (1.9%) 27 (4.0%) 0
248
S. Iwata et al. / Clinica Chimica Acta 264 (1997) 245 – 250
Fig. 1. Frequency distribution of glycosaminoglycan excretion by 6-month-old infants (n 5 10 000). The 95% confidence interval of this population was 71.0–465.5 mg of GAG / g of creatinine.
3.2. Influence of paper diapers The effect of paper diapers on the DMB method is shown in Table 2. The GAG value for the control sample was 27 mg of GAG / g of creatinine, and the GAG values for urine samples squeezed from paper diapers were much higher. When the samples were assayed by the CPC / carbazole method, little difference was seen in the GAG / creatinine values between test samples and controls. The acrylic acid polymer used in the diapers gave a positive reaction (Table 2b), while tissue paper (Table 2a) and acrylic acid (Table 2b) had little effect on the assay.
4. Discussion Although urine samples are useful for metabolic screening, they often contain various substances that interfere with the results of the test. In our mass screening study for MPS, it was found that a component of paper diapers, when mixed with urine, interferes with the DMB method, and this component was
S. Iwata et al. / Clinica Chimica Acta 264 (1997) 245 – 250
249
Table 2 Influence of paper diapers on the DMB method a. Paper diaper and tissue paper
Control Paper diaper Paper diaper Paper diaper Paper diaper Paper diaper Paper diaper Tissue paper
A B C D E F
GAG a / CRE (mg / g cre)
Uronic acid / CRE (mg / g cre)
CRE (mg / ml)
27 3603 6119 1432 6830 4504 9182 23
15.3 23.8 16.5 24.7 12.7 19.5 13.1 15.3
0.62 0.61 0.59 0.60 0.60 0.58 0.59 0.61
b. Crosslinked acrylic acid polymer sodium salt and acrylic acid GAG a (mg / l) Crosslinked acrylic acid polymer sodium salt Acrylic acid (mg / ml)
Lot A Lot B 10 100
587.5 257.5 0.7 3.0
a
Absorbance at 520 nm was measured and compared with 100 mg / l standard solutions of chondroitin-6-sulfate.
suggested to be acrylic acid polymer. Whitley et al. [10] found that heparin, a sulfated GAG, is highly reactive in the assay, while other drugs that are likely to be administered to infants produced no interference, except when formulations containing artificial coloring agents were added to the reaction. De Jong et al. [8] found that urinary protein negatively interfered with the DMB assay at almost physiological concentrations. Positive interference by acrylic acid polymer derived from the paper diaper has not been reported. In a third screening, the 27 samples in our pilot study that had higher GAG values were re-tested using the CPC / carbazole method, and all samples fell within the normal reference range. Since it was found that the paper diapers and acrylic acid polymer do not positively interfere with the CPC / carbazole method, it is desirable to check the false positive results on the DMB method. Although the screened infants have not reached or passed the age at which clinical symptoms become apparent, none of the infants screened so far has become a patient. In conclusion, the DMB method must be considered with caution for use with urine samples collected from paper diapers, since samples that were positive using the DMB method should be re-tested with the CPC / carbazole method.
250
S. Iwata et al. / Clinica Chimica Acta 264 (1997) 245 – 250
Acknowledgments We thank Dr. A. Tanaka (Osaka City University) for technical advice, SANYOKASEI for providing crosslinked acrylic acid polymer sodium salt, and the Gifu Research Center for Public Health, for their cooperation. This study was supported in part by Grants-in-Aid for Scientific Research (08670869) from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Ministry of Health and Welfare, Japan.
References [1] Neufeld EF, Muenzer J. The mucopolysaccharidoses. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease. 7th ed. New York: McGraw-Hill, 1995; 2465–2494. [2] Thomas ED, Ramberg RE, Sale GE et al. Direct evidence for a bone marrow origin of the alveolar macrophage in man. Science 1976;192:1016–8. [3] Hobbs JR. Bone marrow transplantation for inborn errors. Lancet 1981;2:735–9. [4] Hobbs JR, Hugh-Jones K, Barrett AJ, Byron N, Chambers D, Henrry K et al. Reversal of clinical features of Hurler’s disease and biochemical improvement after treatment by bone-marrow transplantation. Lancet 1981;2:709–12. [5] Hugh-Jones K, Hobbs JR, Vellodi A, Hancock M, Sheldon J, Jones S. Long-term follow-up of children with Hurler’s disease treated with bone marrow transplantation. In: Hobbs JR, editor. Correction of Certain Genetic Diseases by Transplantation, 1989. London: Cogent, 1989; 103–111. [6] Bitter T, Muir HM. A modified uronic acid–carbazole reaction. Anal Biochem 1962;4:330– 4. [7] Huang KC, Sukegawa K, Orii T. Screening test for urinary glycosaminoglycans and differentiation of various mucopolysaccharidoses. Clin Chim Acta 1985;151:147–56. [8] De Jong JGN, Wevers RA, Liebrand-van Sambeek R. Measuring urinary glycosaminoglycans in the presence of protein: An improved screening procedure for mucopolysaccharidoses based on dimethylmethylene blue. Clin Chem 1992;38:803–7. [9] Bonsnes RW, Taussy HH. On the colorimetric determination of creatinine by the Jaffe reaction. J Biol Chem 1945;158:581–91. [10] Whitley CB, Ridnour MD, Draper KA, Dutton CM, Neglia JP. Diagnostic test for mucopolysaccharidosis. I. Direct method for quantifying excessive urinary glycosaminoglycan excretion. Clin Chem 1989;35:374–9.
Short communication
Mass screening test for mucopolysaccharidoses using the 1,9-dimethylmethylene blue method: Positive interference from paper diapers a, a a a Shoko Iwata *, Kazuko Sukegawa , Toshiya Sasaki , Mie Kokuryu , Sumiko Yamasita b , Akio Noma b , Shinichi Iwasa c , Naomi Kondo a , Tadao Orii a , d a
Department of Pediatrics, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500, Japan b Clinical Laboratory, Gifu University Hospital, 40 Tsukasa-machi, Gifu 500, Japan c Iwasa Hospital, 161 -1 Nagara Fukumitsu, Gifu 502, Japan d Chubu Gakuin University, 4909 -3 Kurachi, Seki, Gifu 501 -32, Japan Received 25 July 1996; received in revised form 1 April 1997; accepted 15 April 1997
Keywords: Mucopolysaccharidosis; 1,9-Dimethylmethylene blue; Mass screening; Glycosaminoglycans
1. Introduction The mucopolysaccharidoses (MPS) are a group of disorders caused by a deficiency in the lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs). The undegraded GAGs are stored in lysosomes and subsequently are excreted in the urine. Quantitative measurement of urinary GAGs is therefore available for diagnosis of the MPS. The clinical manifestations of the MPS are coarse facies, skeletal deformities, hepatosplenomegaly, joint stiffness and variable degrees of psychomotor developmental delay [1]. Bone marrow transplantation (BMT) has been proposed as a treatment for MPS, both to correct the bone marrow-derived cells in the recipient by
*Corresponding author. Tel.: 81-58-265-1241 ext. 2819; fax: 81-58-265-9011. 0009-8981 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved. PII S0009-8981( 97 )00084-3
246
S. Iwata et al. / Clinica Chimica Acta 264 (1997) 245 – 250
replacement, and also to correct other cells which take up enzyme secreted by the bone marrow-derived cells, especially macrophages [2]. Mass screening for MPS in early infancy could provide a method for presymptomatic diagnosis, and it could realize early BMT, in which clinical outcome would be better than in BMT after clinical manifestation became apparent [3–5]. We have proposed organizing mass screening for MPS in Japan and a pilot study is in progress in the Gifu Prefecture using the dimethylmethylene blue (DMB) method. In this study, we have observed new positive interference from paper diapers. We describe our findings in the pilot study of mass screening for MPS.
2. Materials and methods
2.1. Collection of urine specimens Urine samples were collected from 44 060 infants at 6 months of age. A plastic sheet was placed on the diaper and a piece of cotton was put on it. Such a diaper was attached to the infant and, after the cotton was dampened with urine, it was removed. The urine sample was squeezed from the cotton and was placed in a plastic bottle. It was returned by mail and stored at 2 208C until assay. Informed consent was obtained from the parents of the infants studied.
2.2. Pilot study for mucopolysaccharidoses in the Gifu Prefecture Over a three-year period from 1993–1995, urine samples from 6-month-old infants were screened. Initial screening was carried out using the DMB method. At first, the upper cut-off point was selected arbitrarily and was set at 400 mg of GAG / g of creatinine. The current limit was selected in the light of experience gained from the initial part of the prospective trial. When the value of urinary GAGs was over the cut-off point, a second sample was re-tested by the same method. After the second test, a third assay was carried out with the same samples using the cetylpyridinium chloride (CPC) / carbazol method and monodimensional electrophoresis [6,7].
2.3. Influence of paper diapers To study the influence of a paper diaper on the DMB reaction, the products from six manufacturers were analyzed. A 400-ml volume of urine was absorbed on the paper diaper, incubated at 378C for 12 h, squeezed and assayed by the DMB method. As a control sample, urine was incubated at 378C for 12 h and
S. Iwata et al. / Clinica Chimica Acta 264 (1997) 245 – 250
247
assayed by the DMB method. Since crosslinked acrylic acid polymer sodium salt (acrylic acid polymer) is used as the absorbent of the paper diaper, we analyzed acrylic acid polymer as a candidate of positive interference on the DMB method. A 1-g amount of crosslinked acrylic acid polymer sodium salt (SANYOKASEI, Kyoto, Japan) was dissolved in 200 ml of physiological saline, incubated at 378C for 12 h, filtered and the eluate was assayed by the DMB method. Tissue paper and acrylic acid were used as references.
2.4. Measurement of urinary GAG The DMB and cetylpyridinium chloride (CPC) / carbazole-borate assays were carried out using a modification of the method of de Jong et al. [8] and of Bitter and Muir [6], respectively. Urinary creatinine was measured by the Jaffe method [9]. The measurement was automatically performed with a MR 5000 plate reader (Dynatech, USA).
3. Results
3.1. Mass screening pilot study for MPS We tested 44 060 infants at 6 months of age. With an upper cut-off limit of 400 mg of GAG / g of creatinine, 842 infants (1.9%) were positive. A second sample was requested for the positive individuals, of which 671 were re-tested: In the second screening, 27 cases were found to be positive and in the third screening using the CPC / carbazole method, all specimens were found to be negative (Table 1). To determine the range of GAG excretion in this population, we analyzed 10 000 samples obtained from presumed-normal 6-month-old infants using the DMB method. By a non-parametric method, the 95% confidence interval of this population was 71.0–465.5 mg of GAG / g of creatinine (Fig. 1).
Table 1 Pilot mass screening study for MPS in the Gifu Prefecture
First screening Second screening Third screening
n
Positive (%)
44 060 671 27
842 (1.9%) 27 (4.0%) 0
248
S. Iwata et al. / Clinica Chimica Acta 264 (1997) 245 – 250
Fig. 1. Frequency distribution of glycosaminoglycan excretion by 6-month-old infants (n 5 10 000). The 95% confidence interval of this population was 71.0–465.5 mg of GAG / g of creatinine.
3.2. Influence of paper diapers The effect of paper diapers on the DMB method is shown in Table 2. The GAG value for the control sample was 27 mg of GAG / g of creatinine, and the GAG values for urine samples squeezed from paper diapers were much higher. When the samples were assayed by the CPC / carbazole method, little difference was seen in the GAG / creatinine values between test samples and controls. The acrylic acid polymer used in the diapers gave a positive reaction (Table 2b), while tissue paper (Table 2a) and acrylic acid (Table 2b) had little effect on the assay.
4. Discussion Although urine samples are useful for metabolic screening, they often contain various substances that interfere with the results of the test. In our mass screening study for MPS, it was found that a component of paper diapers, when mixed with urine, interferes with the DMB method, and this component was
S. Iwata et al. / Clinica Chimica Acta 264 (1997) 245 – 250
249
Table 2 Influence of paper diapers on the DMB method a. Paper diaper and tissue paper
Control Paper diaper Paper diaper Paper diaper Paper diaper Paper diaper Paper diaper Tissue paper
A B C D E F
GAG a / CRE (mg / g cre)
Uronic acid / CRE (mg / g cre)
CRE (mg / ml)
27 3603 6119 1432 6830 4504 9182 23
15.3 23.8 16.5 24.7 12.7 19.5 13.1 15.3
0.62 0.61 0.59 0.60 0.60 0.58 0.59 0.61
b. Crosslinked acrylic acid polymer sodium salt and acrylic acid GAG a (mg / l) Crosslinked acrylic acid polymer sodium salt Acrylic acid (mg / ml)
Lot A Lot B 10 100
587.5 257.5 0.7 3.0
a
Absorbance at 520 nm was measured and compared with 100 mg / l standard solutions of chondroitin-6-sulfate.
suggested to be acrylic acid polymer. Whitley et al. [10] found that heparin, a sulfated GAG, is highly reactive in the assay, while other drugs that are likely to be administered to infants produced no interference, except when formulations containing artificial coloring agents were added to the reaction. De Jong et al. [8] found that urinary protein negatively interfered with the DMB assay at almost physiological concentrations. Positive interference by acrylic acid polymer derived from the paper diaper has not been reported. In a third screening, the 27 samples in our pilot study that had higher GAG values were re-tested using the CPC / carbazole method, and all samples fell within the normal reference range. Since it was found that the paper diapers and acrylic acid polymer do not positively interfere with the CPC / carbazole method, it is desirable to check the false positive results on the DMB method. Although the screened infants have not reached or passed the age at which clinical symptoms become apparent, none of the infants screened so far has become a patient. In conclusion, the DMB method must be considered with caution for use with urine samples collected from paper diapers, since samples that were positive using the DMB method should be re-tested with the CPC / carbazole method.
250
S. Iwata et al. / Clinica Chimica Acta 264 (1997) 245 – 250
Acknowledgments We thank Dr. A. Tanaka (Osaka City University) for technical advice, SANYOKASEI for providing crosslinked acrylic acid polymer sodium salt, and the Gifu Research Center for Public Health, for their cooperation. This study was supported in part by Grants-in-Aid for Scientific Research (08670869) from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Ministry of Health and Welfare, Japan.
References [1] Neufeld EF, Muenzer J. The mucopolysaccharidoses. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease. 7th ed. New York: McGraw-Hill, 1995; 2465–2494. [2] Thomas ED, Ramberg RE, Sale GE et al. Direct evidence for a bone marrow origin of the alveolar macrophage in man. Science 1976;192:1016–8. [3] Hobbs JR. Bone marrow transplantation for inborn errors. Lancet 1981;2:735–9. [4] Hobbs JR, Hugh-Jones K, Barrett AJ, Byron N, Chambers D, Henrry K et al. Reversal of clinical features of Hurler’s disease and biochemical improvement after treatment by bone-marrow transplantation. Lancet 1981;2:709–12. [5] Hugh-Jones K, Hobbs JR, Vellodi A, Hancock M, Sheldon J, Jones S. Long-term follow-up of children with Hurler’s disease treated with bone marrow transplantation. In: Hobbs JR, editor. Correction of Certain Genetic Diseases by Transplantation, 1989. London: Cogent, 1989; 103–111. [6] Bitter T, Muir HM. A modified uronic acid–carbazole reaction. Anal Biochem 1962;4:330– 4. [7] Huang KC, Sukegawa K, Orii T. Screening test for urinary glycosaminoglycans and differentiation of various mucopolysaccharidoses. Clin Chim Acta 1985;151:147–56. [8] De Jong JGN, Wevers RA, Liebrand-van Sambeek R. Measuring urinary glycosaminoglycans in the presence of protein: An improved screening procedure for mucopolysaccharidoses based on dimethylmethylene blue. Clin Chem 1992;38:803–7. [9] Bonsnes RW, Taussy HH. On the colorimetric determination of creatinine by the Jaffe reaction. J Biol Chem 1945;158:581–91. [10] Whitley CB, Ridnour MD, Draper KA, Dutton CM, Neglia JP. Diagnostic test for mucopolysaccharidosis. I. Direct method for quantifying excessive urinary glycosaminoglycan excretion. Clin Chem 1989;35:374–9.