Review Paper - Current Pediatric Research (2017) Volume 21, Issue 3

Methodology used for collecting saliva of preterm newborns to measure cortisol: Systematic review

¹ PhD Student, Child and Adolescent Health, Department of Pediatrics, Faculty of Medical Sciences, University of Campinas, UNICAMP, São Paulo, Brazil

²Assistant Professor, Division of Neonatology, Department of Pediatrics, Faculty of Medical Sciences, University of Campinas, UNICAMP, São Paulo, Brazil.

³Deparment of Pathology, Medical Science School, University of Campinas, UNICAMP, São Paulo, Brazil.

⁴Professor, Pediatrics Neonatal Section, Department of Pediatrics, University of Campinas, UNICAMP, Sao Paulo, Brazil.

*Corresponding Author:

Vanessa Carina Pepino Stelini
Rua Adolfo Barbalho de Uchoa Cavalcanti
131, casa 24, cep: 13092-200. Jardim Lumen Christ
Campinas-SP
Brazil.
Tel: 55 19 9 96481049
E-mail: vanpepino@yahoo.com

Accepted date: June 05, 2017

Abstract

Objective: Systematically analyze the methods used by clinical trials published in the last 10 years to collect preterm newborn saliva to measure cortisol. Method: Three databases PubMed, Scopus and Embase were searched in December 2016. Two researchers independently selected studies by title, and the abstracts of the selected studies were read to determine which ones met the inclusion criteria. All clinical trials of hospitalized preterm newborns that collected saliva to measure salivary cortisol were included. Results: Two types of methods were used for collecting saliva: aspiration or absorption, using absorbent materials. All nine aspiration-based studies and the 24 absorptionbased studies managed to measure salivary cortisol in at least some samples. Twenty-one studies reported losing some samples. The most common reasons for sample losses were insufficient saliva. Meta-regression (meta-analysis) and random effects model investigated possible relationships between percentage of sample losses and the following variables at a significance level of 5%: collection method, collection material, collection time and type of assay. Aspiration resulted in fewer sample losses than absorption (p<0.0001). However, only 33.3% (3 of 9) of aspiration-based studies reported the number of sample losses against 81.8% (18 of 22) of absorption-based studies. The most used assays to measure salivary cortisol were ELISA and radioimmunoassay. Conclusion: Salivary cortisol measurement in preterm newborns may be useful for assessing and comparing the level of stress generated or relieved by different stimuli. Saliva collection methods have not been standardized, preventing the reproduction of studies that use these methods.

Keywords

Salivary cortisol, Preterm, newborn.

Introduction

Although the inherent care provided by a neonatal intensive care unit (NICU) stresses preterm newborns (PTNs), it is critical for their survival [1]. Studies have searched for alternative and complementary therapies, such as music therapy, tactile-kinesthetic stimulation, kangaroo care, and skin-to-skin contact, to the standard treatment to reduce factors that cause tension in an NICU [2-5]. Salivary cortisol level, which can be detected already in the first week of life, increases in individuals experiencing adverse situations, and PTNs have higher salivary cortisol level than term newborns [6,7]. In order to assess the efficacy of alternative and complementary therapies, noninvasive tests, such as PTN salivary cortisol measurement, have been performed [8-12]. Noninvasive methods that measure cortisol to possibly determine newborns’ level of discomfort may be used to assess routine and new NICU procedures.

Despite the possible benefits of this type of assessment in newborns, studies discuss whether salivary cortisol is indeed associated with newborn’s stress and plasma cortisol levels [13,14]. Studies that performed salivary cortisol measurements used different methodologies. The objective of this review was to systematically analyze the methods used by clinical trials published in the last 10 years to collect PTNs’ saliva to measure cortisol, to determine the most successful methods for collecting saliva and to list the advantages and disadvantages of each method.

Method

Three databases, PubMed (United States National Library of Medicine of the National Institutes of Health), Scopus, and Embase (Excerpta Medica Database) were searched in December 2016 using the keywords “salivary cortisol” AND “premature.” This was the combination of keywords that returned the highest number of studies in each database.

A database was created using the software EndNote, which manages references, and the data were judiciously stored in folders. Two researchers independently selected studies by title and the abstracts of the selected studies were read to determine which ones met the inclusion criteria. If the researchers disagreed, the study was saved to be read in full.

Selection Criteria

All studies found in the abovementioned databases that collected saliva from PTNs were selected.

Inclusion Criteria

All clinical trials of hospitalized PTNs that collected saliva to measure salivary cortisol published in the last 10 years in Portuguese, English or Spanish were included.

Exclusion Criteria

Studies that collected saliva after the newborn left the hospital, posters, studies that measured cortisol in term newborns, and studies in languages other than those mentioned above were excluded.

Results

The keywords “salivary cortisol” AND “premature” returned 235 studies, 86 from PubMed, 69 from Embase and 80 from Scopus. Repeated studies were eliminated, resulting in 152 studies for title assessment. Two researchers performed all inclusion and exclusion procedures independently.

Forty-five articles were excluded based on their titles, and later, another 35 studies were excluded based on their abstracts. Twenty-one studies of the remaining 72 were excluded because they had been published before December 2006. Thus, 51 studies were read in full, of which 32 complied with the inclusion and exclusion criteria. Nineteen studies were eliminated during this phase for the following reasons: five did not collect saliva during hospital stay, four did not collect saliva from PTNs, two did not measure salivary cortisol, only plasma cortisol, three were presented as posters, two only collected maternal saliva (n=2), one was published in Persian, and the whole text of two was not found (Figure 1) [15-33]. One more study listed in the references of one of the reviewed studies was included [13]. In summary, 235 articles were found in the three researched databases, and after elimination of the duplicates and application of the inclusion and exclusion criteria, 33 studies remained (Table 1).

Figure 1: Flowchart

Table 1. Description of study objectives.

Two types of methods were used for collecting PTNs’ saliva: aspiration or absorption, using absorbent materials. Aspiration was performed with a pipette, syringe connected to a plastic tube, needleless push button blood collection set or probe or tracheal suction set [8,9,11,34-40]. The absorbent materials used for absorbing the newborns’ saliva were swabs or cotton buds of different brands and models, filter paper or sterile dental cotton roll tied with a dental floss [6,10-14,37,41-57]. One of the studies used both collection methods, swab and aspiration and one did not specify how PTN saliva was collected [37,58].

Only one of the studies that specified saliva extraction method did not use centrifugation. That study collected saliva with a sterile dental cotton roll and extracted the saliva by placing the roll inside a 5 ml syringe and pressing the plunger to expel the saliva [57].

Collection time varied between studies. Aspiration-based studies did not inform the duration of the procedure, and only two studies reported the amount of collected saliva [35,39]. Aspiration pressure was not informed by any study, and only one study reported using “enough negative pressure to suck 0.5 to 1.0 ml of newborn saliva” [38]. Nine absorption-based studies did not inform for how long the absorbent material stayed in the newborn’s mouths [2,6,10,14,37,41,47,48,53]. Four studies that provided such information left the material in the newborns’ mouths for 5 min [45,50,52,54]; one, for 10 min [43]; one, for at least 20 min [51]; one, for 5 to 10 min [42]; one, for 20 to 25 min [44]; one, for 1 to 2 min [46]; one, for 30 to 60 s [49]; and one left the first 10 samples in the PTNs’ mouths for 3 min and the subsequent 53 samples, for 20 min [13].

All nine aspiration-based studies and the 24 absorptionbased studies managed to measure salivary cortisol in at least some samples. Two studies omitted the results because their samples did not have enough saliva [37,43].

Twenty-one studies reported losing some samples [6,8,10-14,35,37,41-46,48,51,53,55-57]. The most common reasons for sample losses were insufficient saliva and/or contamination with blood [6,8,10-14,37,41- 46,48,51,53,55-57]. Only one study lost samples due to the viscosity of the absorbent material [35]. The percentage of sample losses due to insufficient saliva ranged from 0% to 90%. Of the studies that specified collection method and reported the total number of sample losses, three used aspiration and 18 used absorbent materials. Six aspirationbased studies and four absorption-based studies did not inform whether any samples were lost or the number of sample losses (Table 2).

Table 2. Description of collection method, material, assay and % of sample lost.

This group of studies (three that used aspiration and 18 that used absorbent materials) made 181 and 3560 attempts to collect saliva using aspiration and absorbent materials, respectively. Aspiration-based studies lost fewer samples than absorption-based studies, regardless of assay method. The measures of central tendency of the percentage of aspiration-based sample losses were 15.19% (mean) and 11.66% (median), and of absorption-based sample losses were 23.91% (mean) and 15.92% (median). However, only 33.3% (3 of 9) of aspiration-based studies reported the number of sample losses against 81.8% (18 of 22) of absorption-based studies.

Meta-regression (meta-analysis) and random effects model investigated possible relationships between percentage of sample losses and the following variables at a significance level of 5%: collection method, collection material, collection time, and type of assay (Table 3). Aspiration resulted in fewer sample losses than absorption (p<0.0001).

Table 3. Evaluation of % loss according to the studied factors (type, material, essay and collection time) - Meta regression

The studies used the following assays to measure salivary cortisol: radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), liquid chromatographymass spectrometry, electrochemiluminescence and fluorescent immunoassay [6,8-14,34,36-39,41-57].

Inspection of the oral mucosa before saliva collection was one of the measures taken by the studies to avoid sample contamination with blood [46]. Three studies collected saliva 30 to 60 minutes after feeding to avoid sample contamination with milk [6,8,9,11,12,35,39,40,42,46- 48,57]. Two studies cleaned the oral cavity before saliva collection, and one study cleaned the oral cavity and collected a second saliva sample if the first sample appeared contaminated with blood [35,45,57]. Only one study did not exclude samples contaminated with blood or milk [2].

Discussion

The present review, which included studies published in the last ten years, aimed to help future studies by describing saliva collection methods, collection time, collection materials and the success rates of each method. The objective of most reviewed studies was to compare the level of stress in PTNs undergoing different therapies (Table 1). Salivary cortisol measurement may be a useful tool for assessing the cost-benefit ratio of some relaxation methods and the effects of stress caused by the NICU routine (heel lance, diaper change) [6,9,34,41].

The main objective of Ng et al. [45] was to describe a new methodology to collect PTNs’ saliva to measure cortisol. Although saliva collection is generally regarded as noninvasive, the local research ethics committee that approved said study considered suction invasive as it could injure PTN mucous membranes, which are extremely fragile, and contaminate the sample with blood. Another possible disadvantage of aspiration has to control suction pressure. None of the reviewed studies described the pressure parameters used. However, 9 of the 33 reviewed studies used aspiration, and none reported complications, such as mucosal injury during the procedure. On the other hand, two of the three studies that excluded samples contaminated with blood were aspiration based [8,11]. Unlike suction, the method described by Ng et al. [45] used swabs, which was well tolerated by PTNs as they did not demonstrate any sign of discomfort during collection. The authors found no adverse effects and 85% of the samples contained an adequate amount of saliva [46].

Absorbent material was the method of choice for saliva collection, used by 23 of the 33 reviewed studies. The materials included cotton buds, pediatric swabs or eye sponges of various brands [10,14,44,45]. The authors of two studies split the swab in four parts or in half, attaching it to a cotton bud to reduce its size and fit more comfortably in the PTNs’ mouths [2,49]. Other absorbent materials used by the studies were filter paper and sterile dental cotton rolls [13,55,56,57].

Morelius et al. [58] assessed plastic and wooden shaft swabs immediately after collection, 24 h after collection and 48 h after collection. Mean salivary cortisol was significantly lower in wooden shaft swabs 24 h (40%, p 0.001) and 48 h (49%, p 0.001) after collection than in wooden shaft swabs immediately after collection. However, the cortisol levels in samples collected with plastic shaft swabs did not differ. Hence, the authors recommend not to use both types of swabs in the same trial and to avoid using wooden shaft swabs as wood may absorb cortisol [59].

Twenty-one studies lost some samples due to insufficient saliva and/or blood contamination. Sometimes collecting an adequate amount of saliva is a challenge as the mucosa of some PTNs is extremely dry, especially in PTNs receiving anticholinergic agents, that is, mydriatic agents [42,53]. There is also loss caused by evaporation while the sample is frozen for storage or thawed for analysis [42].

Most studies used centrifugation to extract saliva from the absorbent materials. Such studies used Eppendorf® tubes or Salimetrics® tubes, which contain a separate compartment to store saliva and keep it separated from the absorbent material after centrifugation [2,10,41,42]. Ashwood et al. [56] used sterile dental cotton rolls to absorb saliva and then extracted the saliva by placing the roll inside a 5 ml syringe barrel and pressing the plunger top.

Inadequate saliva volume for the assay, the most common cause of sample loss, may be directly related to the type of assay chosen to measure cortisol as different assays require different amounts of sample. Studies that use assays that require smaller volumes are less likely to lose samples due to inadequate saliva volume. An advantage of using aspiration is the possibility of placing the saliva in a graduated container, which allows immediate awareness of the amount collected. Thus, collection time may be increased until the necessary amount is collected. On the other hand, saliva collected by absorbent materials can only be quantified after centrifugation or extraction by some other means, which is usually done some time after collection. None of the reviewed aspiration-based studies reported collection time while 68.2% (15 out of 22) of the absorption-based studies did (Table 2).

Some studies have not found an association between plasma and salivary cortisol levels, but other studies have [7,10,45,49,50]. Regardless of this correlation, salivary cortisol increases in individuals submitted to stressful situations [7]. Moreover, almost all studies reviewed herein managed to measure cortisol in at least part of the samples, regardless of saliva collection method, suggesting that this procedure is useful but not yet standardized.

Limitations

Although three databases were searched, this review may have missed studies compliant with the inclusion and exclusion criteria. The data in some reviewed studies may have been misinterpreted as they were not clear.

This review only included studies published in the last 10 years because collection materials have changed significantly and many studies were published during this period. Thus, earlier studies may contain important data not included herein.

Conclusion

Salivary cortisol measurement in preterm newborns may be useful for assessing and comparing the level of stress generated or relieved by different stimuli. Saliva collection methods have not been standardized, preventing the reproduction of studies that use these methods.

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