Patients
This was a prospective cross-sectional study on children with short stature or decreased growth velocity who were examined at Aichi Medical University Hospital between April 2015 and March 2020. We used the following inclusion criteria: (a) referred to Aichi Medical University for the evaluation of short stature or decreased growth velocity; (b) short stature of ≤ −2 SD or height velocity of ≤ −1.5 SD in >2 years below the mean for sex and age [10]; and (c) >1 year of age and before the completion of puberty, according to Tanner stages. The exclusion criteria were the presence of recognized congenital abnormalities, such as hypothyroidism; small for gestational age; Turner’s syndrome; and trisomy 21.
General biochemical tests, thyroid function test, bone age, and IGF-1 were examined before GH secretion test in consecutive patients who met the inclusion criteria. The radius, ulna, and short bone method was used for evaluating bone age [11]. The patients were divided into GH and non-GH groups according to the response to the GH secretion test. In Japan, GHD is diagnosed when the peak GH is ≤ 6.0 ng/mL in two GH secretion tests [9]. A cutoff of 6 ng/mL was determined by the Japanese National Health Insurance program. Stimulation tests using clonidine, arginine, and L-dopa were performed in that order, using the algorithm shown in Figure 1. GHD was diagnosed if the GH peak levels were < 6 ng/mL in the two stimulation tests. If the GH peak was above the cutoff level in the clonidine stimulation test, the next stimulation test was not performed. If the GH peak of the arginine stimulation test was 6–8 ng/mL, the third L-dopa stimulation test was performed. If the GH peak of the arginine stimulation test was > 8 ng/mL, the third test was not performed as GHD was unlikely to be present. Glucagon and insulin were not used in this study because glucagon requires a long examination time of 180 min and insulin results in an adverse effect of severe hypoglycemia.
After overnight fasting, the stimulation test was started at 6:30 for children <6 years old and at 9:00 for those >6 years old because of fasting tolerance. Sampling was done at 0, 30, 60, 90, and 120 minutes. Clonidine (5 µg/kg), arginine (10 mg/kg), and L-dopa (10 mg/kg) were administered as the stimuli for the GH secretion test. After the diagnosis of GHD, head MRI was performed before starting GH replacement therapy.
Hormone assays
Serum IGF-1 was measured by electrochemiluminescence immunoassay (Elecsys IGF-1; Roche Diagnostics, Tokyo, Japan), which was calibrated against the WHO International Standard 02/254. The values of serum IGF-1 were transformed into SDs, according to the established reference ranges of the assay for sex and calendar age [12]. GH was measured by immunoenzymometric assay (E Test TOSOH II HGH; Tosoh Co., Ltd., Tokyo, Japan), which was standardized against the WHO International Standard 98/574.
Statistical analysis
We calculated point estimates for IGF-1 (SD) sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic efficiency (DE), positive likelihood ratio (PLR), and negative likelihood ratio (NLR) for predicting the presence of GHD. Data were shown as median (interquartile range) for chronological and bone age, and as mean ± SD for the other numerical variables. Based on the f-test, Student’s t-test was performed in the case of homoscedasticity and the Mann–Whitney U test was performed in the case of unequal variances to compare the IGF-1 level and other variables between the two groups. Spearman’s rank correlation coefficient test was performed to investigate the relationship of IGF-1 (SD) with age, bone age, height (SD), target height (SD), height velocity before examination (SD), weight (SD), body mass index (BMI) (SD), and maximum peak GH (ng/mL). Correlation was defined as very weak if <0.2, weak if ≥0.2 and <0.4, moderate if ≥0.4 and <0.6, strong if ≥0.6 and <0.8, and very strong if ≥0.8. Receiver operating characteristic (ROC) analysis with the Youden index was used to compare the discriminatory performances of IGF-1 in the diagnosis of GHD. Based on the area under the ROC curve (AUC), performance was considered as acceptable if >0.7 and ≤0.8 and excellent if >0.8.
All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) [13], which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R commander designed to add statistical functions frequently used in biostatistics.