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Original Paper Received: March 1, 2002 Accepted after revision: November 20, 2002 Horm Res 2003;59:229–233 DOI: 10.1159/000070222 GH Values after Clonidine Stimulation Measured by Immunofluorometric Assay in Normal Prepubertal Children and GH-Deficient Patients Eveline G.P. Silva Natasha Slhessarenko Ivo J.P. Arnhold Marcelo C. Batista Vivian Estefan Maria G.F. Osorio Suemi Marui Berenice B. Mendonca Unidade de Endocrinologia do Desenvolvimento e Laboratório de Hormônios e Genética Molecular LIM /42, Hospital das Clı́nicas da Faculdade de Medicina da Universidade de São Paulo, Brazil Key Words GH peak W Clonidine W Immunofluorometric assay Abstract Objective: To establish the cut-off values of GH measured by immunofluorometric assay, a more sensitive and specific assay, in normal prepubertal children and compare their values with those of proven GH-deficient patients. Methods: 30 normal children (20 males) and 26 patients with known causes of GH deficiency were submitted to the clonidine test and their GH values were compared. A powdered clonidine tablet (0.1 mg/m2) was given orally and blood samples for GH measurements were drawn at times –30, 0, 60, 90 and 120 min. Results: GH peak values presented a wide variation ranging from 1.7 to 25 Ìg/l (mean B SD = 12.87 B 5.8 Ìg/l) in the normal group. The cut-off values for the 5th and 10th percentiles of the distribution curve were 3.3 and 5.5 Ìg/l, respectively. In the GH deficiency group, maximum GH levels after clonidine stimulation ranged from ! 0.1 to 2.1 Ìg/l (0.56 B 0.58 Ìg/l). Conclusions: The cut-off values obtained with the immunofluorometric method are lower than the ones obtained by radioimmunoassay. We ABC © 2003 S. Karger AG, Basel 0301–0163/03/0595–0229$19.50/0 Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com Accessible online at: www.karger.com/hre suggest a cut-off value of 3.3 Ìg/l (5th percentile) that ensures 100% of sensitivity along with 93% of specificity to exclude the diagnosis of GH deficiency when using this immunofluorometric method. Copyright © 2003 S. Karger AG, Basel Introduction Classically, GH deficiency (GHD) is diagnosed biochemically after at least two stimulation tests, with GH values !7 or !10 Ìg/l measured by radioimmunoassay (RIA) using polyclonal antibodies [1–12]. The development of monoclonal antibodies, non-isotopic assays and the use of more accurate reference standards contributed to the large variability of the results obtained by different assays [13, 14]. Therefore, each laboratory must establish its own cut-off values for each method employed [15, 16]. We measured GH by immunofluorometric assay (IFMA) after clonidine stimulation test in 30 normal prepubertal children in order to establish cut-off values for a normal GH response and applied these values in the diagnosis of GHD. Berenice B. Mendonca, MD Disciplina de Endocrinologia, Hospital das Clı́nicas da Universidade de São Paulo Av Dr Eneas de Carvalho Aguiar, 155, 2 andar, Bloco 6, PAMB São Paulo, SP 05403-900 (Brazil) Tel. +55 11 3062 6654, Fax +55 11 3083 0626, E-Mail [email protected] Table 1. Clinical features of normal and GH-deficient subjects Groups n CA years Gender Height (SD) WHI, % Normal prepubertal 30 2.5–10.81 20 M/10 F GH-deficient 26 1.8–22.5 16 M/10 F –0.57B0.771 (–1.9 to 0.82) –4.6B1.42 (–7.7 to –2.0) 96.4B7.72 85U117 100B14 (82U130) CA = Chronological age; WHI = weight for height index = weight/weight for height age !100. 1 Mean B SD (range in parentheses). Subjects and Methods The present study was approved by the Ethics Committee of Hospital das Clı́nicas da Universidade de São Paulo and the samples were collected after written consent was obtained from parents or tutors. The clinical data of all individuals are shown in table 1. The control group consisted of 30 healthy prepubertal children with normal height (within mean B 2 SD) for chronological age and appropriate weight for height age (WHI: 80–120). All GH-deficient patients were also at prepubertal Tanner stage. All patients had congenital GHD: 12 had isolated GHD and 14 had multiple hypothalamic-pituitary hormone deficiencies. Eight patients had molecular defects in the GH, GHRHR or PROP1 genes and the others had an ectopic posterior pituitary lobe, or pituitary stalk interruption on MRI. The clonidine stimulation test consisted in the collection of two basal blood samples and 60, 90 and 120 min after the oral intake of powdered clonidine tablets (0.1 mg/m2) dissolved in 5 ml of water to avoid difficulties in clonidine absorption. The test was performed when thyroid function was normal and was well tolerated. All patients presented drowsiness 60 min after clonidine intake and slight fall in blood pressure was also observed. All but 5 GH-deficient subjects were also submitted to insulin tolerance test (ITT). The ITT consisted in the collection of two basal blood samples and 15, 30, 45, 60 and 90 min after an intravenous injection of insulin (0.05–0.1U/ kg). All patients presented clinical signs of hypoglycemia confirmed by a glucose level ! 40 mg/dl. GH was measured by immunofluorometric kits (Delfia® kit, Wallac, Turku, Finland) based on the direct sandwich technique using two monoclonal antibodies specific for the 22-kDa form. The assays were standardized with the international standard 80/505 of the World Health Organization. The detection limit of this assay was 0.01 Ìg/l and the intra-assay coefficient of variation was lower than 6% in the working range of the standard curve (0.1–36 Ìg/l). IGF-I was measured in plasma after extraction with ethanol 87.5% and HCl 2 N 12.5%, by RIA, using kits from Nichols Institute (San Juan Capistrano, Calif., USA) in all individuals. Cross-reaction with IGF-II and proinsulin was referred to be lower than 2 and 0.1%, respectively. IGFBP-3 was measured in serum by immunoradiometric assays using kits from the Diagnostic Systems Laboratories Inc. (DSL, Webster, Tex., USA). No cross-reaction was referred with other binding proteins (IGFBP-1, 2 and 4). The inter-assay variation, as defined by repeated measurements of serum controls run in different assays, was ! 10%, for IGF-I levels from 18 to 500 ng/ml and for IGFBP-3 levels from 0.14 to 12 mg/l. 230 Horm Res 2003;59:229–233 The results were analyzed by the Statistics Program for Windows (version 4.0, Statsoft Inc. 1993). The normality hypothesis was tested over the distribution of peak GH values after stimulation, using the Shapiro-Wilk test. The means of IGF-I and IGFBP-3 and GH peak values obtained from both groups were compared by Student’s t test, and p values ! 0.05 were considered statistically significant. GH values ! 10 ng/ml by IRMA were compared to the ones obtained by IFMA, using Spearman’s correlation test. The cut-off values for GH by immunofluorometric method were taken at the 2.5th, 5th and 10th percentiles of the distribution curve of GH values after clonidine stimulation in normal subjects. The probabilities of false-positive tests for the classic cut-off values in RIA, 7 or 10 Ìg/l, were also analyzed on these curves. The cut-off values for basal IGF-I and IGFBP-3 were taken at –1 SD of the mean values and the analysis of their diagnostic properties was also performed. Results GH values after the clonidine test in the control group are shown as mean, SD, and minimum-maximum values. We observed a wide variation in GH response to clonidine and the lowest GH response in this group was 1.7 Ìg/l (table 2). In the GH-deficient group, the highest GH peak value after clonidine was 2.1 Ìg/l. GH peak value after ITT (n = 21) was not significantly different from GH peak values after clonidine, 0.32 B 0.34 vs. 0.56 B 0.58 Ìg/l, p = 0.09, respectively. The Shapiro-Wilk normality test (W) showed a normal distribution of GH values after stimulation in the control group (W = 0.98; p = 0.95). The cut-off values obtained at the 2.5th, 5th and 10th percentiles of the distribution curve of the control group of 30 subjects were 1.5, 3.3 and 5.5 Ìg/l, respectively. The characteristics of the clonidine test for the diagnosis of GHD were calculated with different cut-off values and are presented in table 3. If the current RIA cut-off values of 7 and 10 Ìg/l were used, it would imply in the diagnosis of GHD in 16.7 and 33.3% Silva/Slhessarenko/Arnhold/Batista/ Estefan/Osorio/Marui/Mendonca Table 2. Basal IGF-I, IGFBP-3 and GH peak values after oral clonidine (0.1 mg/m2) stimulation test in normal and GH-deficient subjects Groups Normal prepubertal GH-deficient IGF-I, ng/ml IGFBP-3, mg/l GH peak, Ìg/l mean B SD mean B SD mean B SD range 116B68 24.5B14* 3.66B1.06 0.97B0.61* 12.9B5.8 0.56B0.58** 1.7–25 !0.1–2.1 5th centile 10th centile 3.3 5.5 * p ! 0.001; ** p ! 0.05 GH-deficient vs. normal subjects. Table 3. GH response to clonidine test data for the diagnosis of GHD by IFMA using several cut-off levels GH cut-off level Ìg/l (centile) Sensitivity % Specificity % 1.5 (2.5th) 3.3 (5th) 5.5 (10th) 7.01 101 85 100 100 100 100 100 93.3 90 83.3 66.7 1 Table 4. Basal IGF-I and IGFBP-3 levels for the diagnosis of GHD using –1 SD as the cut-off levels FPR % FNR % Accuracy % 15 0 0 0 0 93 96 95 91 82 FPR % FNR % Accuracy % 6.7 13.3 0 3.8 0 0 0 6.7 10 16.7 33.3 Classical cut-off values. FPR = False positive rate; FNR = false negative rate. Test IGF-I IGFBP-3 IGF-I + IGFBP-3 Sensitivity % 96.2 100 100 Specificity % 93.3 86.7 100 94.6 92.4 100 FPR = False positive rate; FNR = false negative rate. of the normal individuals, respectively. But if we used the 5th and 10th percentiles of the distribution curve as cutoff values, only 6.7 and 10% of the normal individuals would be classified as GH-deficient. Basal IGF-I and IGFBP-3 values are presented in tables 2 and 4. IGF-1 and IGFBP-3 levels in the GH-deficient group were lower than in the normal one (p ! 0.05). We established as cut-off value –1 SD of the mean obtained from the control group for the diagnosis of GHD (table 4). IGF-I values were low in 2 subjects from the control group (6.7%) and none had low IGFBP-3, whereas in the GH-deficient group, 3 subjects presented IGFBP-3 values higher than –1 SD, and 1 presented IGF-I higher than –1 SD. GH Values after Clonidine Stimulation Discussion Presently, several types of assays for GH measurement are available: RIA, immunoradiometric assay (IRMA), enzyme-immunoassay assay (ELISA) chemoluminescence assay and IFMA [17–22]. The presence of GHbinding proteins [23, 24], the heterogeneity of the circulating GH molecules [14, 24, 25] as well as the different types of antibodies, either monoclonal or polyclonal, could account for the variability observed in the results obtained by these methods. The diagnosis of GHD during childhood and adolescence has been the subject of much controversy [26–28]. To ensure that patients are appropriately identified and treated, the GH Research Society (GRS) held a workshop Horm Res 2003;59:229–233 231 on October 17–21 1999, in Eilat, Israel [26]. One of the conclusions of this workshop was the necessity to establish normative data for GH values using newer monoclonal-based assays and recombinant hGH reference preparations. However, there are limited normative data for such GH assays and more data in normal children are desirable within ethical guidelines. We used an IFMA with monoclonal antibodies specific for the 22-kDa GH form. This method has several advantages, such as being non-radioactive, requiring only small amounts of serum, high sensitivity, accuracy and precision. In a preliminary study performed at our laboratory, Marui et al. [29] compared GH response curves after stimulation (hypoglycemia and clonidine) by the IRMA and IFMA methods, in 9 children with short stature (–1.2 to –3.7 SD for height) and/or slow growth velocity. A significant positive correlation between the two methods (r = 0.899, p! 0.001) was observed, although GH peak values measured by the IFMA method (mean B SD, 9.4 B 4.3 Ìg/l) were lower than the ones for the IRMA method (17 B 8 Ìg/l). However, for the same IFMA values, there were different IRMA values, suggesting that the use of the mathematical correlation (IRMA = 0.575 + 1.58 W IFMA) would not always represent the actual GH value. The cut-off level of GH measured by RIA, used in the diagnosis of GHD, has always been determined arbitrarily as 7 and 10 Ìg/l. If we had used the current GH cut-off values in our normal individuals analyzed by IFMA, it would have implied in the diagnosis of GHD in 17 and 33% of them, respectively. Choosing a cut-off value for a test depends on the knowledge of the distribution curve obtained from normal individuals and patients. According to the basic principles for the test selection, the ideal test is the one that does not overlap results from patients and normal individuals [30]. For the majority of the tests, however, there is some overlapping between these two groups. Statistically, the normal interval comprehends the distribution of 95% of the results and is defined by mean B 2 SD. Therefore, 2.5% of the normal individuals can be found above or below this interval. Choosing a point that presents high specificity, but limited sensitivity, is useful when one wants to confirm a suspected diagnosis, but not as a screening test to rule out a disease. The most appropriate cut-off value must present high sensitivity with a reasonable specificity in order to be used as a screening test. It is difficult to establish a cut-off value with these characteristics for the diagnosis of GHD, since short stature due to GHD has a spectrum of clinical manifestations that can vary from partial or transitory deficiency to complete hor- 232 Horm Res 2003;59:229–233 mone deficiency. When analyzing the different cut-off values obtained from the distribution of GH values in normal subjects (1.5, 3.3 and 5.5 Ìg/l), and the ones classically described in literature, we observed a better performance or accuracy when the values 3.3 and 5.5 Ìg/l were used. Our cases with severe GHD were clearly diagnosed using the cut-off levels at the 5th percentile (3.3 Ìg/l) of the normal group. However, it is possible that patients with partial GHD cannot be identified by this cut-off. Therefore, children with values between 3.3 and 5.5 Ìg/l should be submitted to a second test, mainly those suspected to have partial GHD. In our GH-deficient group, 86.7% of the subjects presented IGFBP-3 values below –1 SD and 96% presented IGF-I below –1 SD. Analyzing together the normal and GH-deficient subjects, the association of IGF-1 and IGFBP-3 levels below –1 SD was found only in the GHdeficient group ensuring a 100% of sensitivity along with 100% of specificity for the diagnosis of GHD (table 4). In a recent study, Attanasio et al. [31] re-evaluated adult-age GH status in 167 patients with GHD diagnosed in childhood. They performed a provocative GH stimulation test (cut-off peak level !3 Ìg/l) and measured basal IGF-I and IGBP-3 (cut-off –2 SDS values using a normal population as reference). They verified that GH peak after stimulation and basal IGF-1 levels presented similar sensitivity (80%) for GHD diagnosis. When IGF-I values and GH response to the test were discordant, IGFBP-3 levels were not useful to establish a diagnosis. Thus, the operational characteristics of a test vary according to the criterion used to define what a normal value is. Therefore, it is important to know such characteristics, not only in order to promote the adequate interpretation of the results, but also to decide which test should be used for a given objective. In conclusion, we determined the normal GH peak values after clonidine stimulation in normal prepubertal children using an IFMA and suggest the use of a cut-off value of 3.3 Ìg/l, which ensures 100% of sensitivity along with 93% of specificity to exclude the diagnosis of GHD by this method. Acknowledgements The authors thank the staff of Laboratório de Hormônios e Genética Molecular LIM/42, particularly Cassia Regina Mazzi, Marcia Ester Paiva Ferreira and Valeria Samuel Lando for technical assistance and Sonia Strong for the English review. 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