## 4 Discussion
Moreover, the procedure took place between 09.00 and 12.00 a.m., since the amount of saliva secretion has been shown to change during the day [29]. As previously mentioned, the passive drooling saliva method obtains many advantages for biomarker assessment, especially compared to blood or serum collection, mostly regarding invasiveness. However, there is no scientific evidence robust enough to support the substitution of one collection method for another [30]. In a recent study where authors examined the potential correlation of the biomarker glutathione in saliva and blood, they found that there was no strong correlation between whole blood and salivary glutathione contents in healthy people [31], thus there is room for research in this field to determine if salivary and blood results obtain the same accuracy in biomarker detection. Enzyme-linked immunosorbent assay (ELISA) is a labeled immunoassay that is considered the gold standard of immunoassay for the detection and quantification of substances such as antibodies, hormones, antigens, proteins, and glycoproteins. In this study, the ELISA method was used for the quantitative analysis of BALP and OC in saliva, since this method has high sensitivity, specificity, and efficiency, and the procedure is simple and cost-effective [32]. Concerning the method of [PERSON] et al. for skeletal growth assessment, intra-examiner as well as inter-examiner reliability was high. It has been shown that this method has poor reproducibility [33], but in the present study, this was not confirmed. Regarding the validation of the CVM method for predicting skeletal maturation age, [PERSON] et al. (2012) implemented a systematic review to determine if the cervical vertebrae maturation method can determine peak pubertal growth reliably [34]. Six studies were of sufficient quality to be included in the analysis. These studies found that the correlation between the CVM method and the hand-wrist method was statistically significant. [PERSON] et al. (2014) [35] carried out a systematic review and meta-analysis on the validity of skeletal maturation assessment by analyzing cervical vertebrae in order to assess whether the technique can replace the use of hand-wrist radiographs to determine peak pubertal growth. This systematic review included nineteen articles that compared radiographs of the hand-wrist and cervical vertebral regions. Positive correlations were observed in all articles and the meta-analysis found a higher correlation in females. The authors concluded that cervical vertebrae maturation indexes show good reliability and can replace hand-wrist radiographs in determining peak pubertal growth. It can be concluded that orthodontic literature has sufficient documentation to prove both methods are almost similar in estimating growth, so the CVM method is a valid method for skeletal growth assessment. The capacity of the ELISA kits to detect protein concentration in a wider range could be regarded as a limitation of the current study. In two patients, the values of BALP and OC were below the detectable range, and they could not be measured. Thus, they were excluded from statistical analyses. Other limitations of the current investigation include the small sample size due to the coronavirus pandemic, as well as the cross-sectional nature of the study. The strengths of the study are the protocol design and the examination of a new biomarker in saliva, which has not been investigated in this biofluid before. In the future, a longitudinal sample to register the variations of the salivary biomarkers over time within the same subjects is needed. Furthermore, another maturation index, such as actual growth by measuring height/weight over time and/or hand-wrist films, is warranted, even though this may not be justified in the early pre-pubertal or post-pubertal phase due to the radiation. In the present study, BALP peak salivary levels seem to correlate with the growth spurt, since higher values were observed in CS3, which were gradually reduced in the following stages.