Elsevier

Clinica Chimica Acta

Volume 413, Issues 13–14, 11 July 2012, Pages 1066-1070
Clinica Chimica Acta

Evaluation of saliva collection devices for the analysis of proteins

https://doi.org/10.1016/j.cca.2012.02.020Get rights and content

Abstract

Background

Human saliva mirrors the body's health and can be collected non-invasively, does not require specialized skills and is suitable for large population based screening programs. The aims were twofold: to evaluate the suitability of commercially available saliva collection devices for quantifying proteins present in saliva and to provide levels for C-reactive protein (CRP), myoglobin, and immunoglobin E (IgE) in saliva of healthy individuals as a baseline for future studies.

Methods

Saliva was collected from healthy volunteers (n = 17, ages 18–33 years). The following collection methods were evaluated: drool; Salimetrics® Oral Swab (SOS); Salivette® Cotton and Synthetic (Sarstedt) and Greiner Bio-One Saliva Collection System (GBO SCS®). We used AlphaLISA® assays to measure CRP, IgE and myoglobin levels in human saliva.

Results

Significant (p < 0.05) differences in the salivary flow rates were observed based on the method of collection, i.e. salivary flow rates were significantly lower (p < 0.05) in unstimulated saliva (i.e. drool and SOS), when compared with mechanically stimulated methods (p < 0.05) (Salivette® Cotton and Synthetic) and acid stimulated method (p < 0.05) (SCS®). Saliva collected using SOS yielded significantly (p < 0.05) lower concentrations of myoglobin and CRP, whilst, saliva collected using the Salivette® Cotton and Synthetic swab yielded significantly (p < 0.05) lower myoglobin and IgE concentrations respectively.

Conclusions

The results demonstrated significantly relevant differences in analyte levels based on the collection method. Significant differences in the salivary flow rates were also observed depending on the saliva collection method. The data provide preliminary baseline values for salivary CRP, myoglobin, and IgE levels in healthy participants and based on the collection method.

Highlights

► The effect of saliva collection methods on the detection of C-Reactive protein (CRP), IgE and mygolbin. ► Significant differences in the salivary flow rates were also observed based on the collection method. ► We report base line values for salivary CRP, myoglobin, and IgE levels in healthy participants.

Introduction

Saliva is an important biological fluid with many functions in the oral cavity including maintaining oral homeostasis [1]. Saliva contains a wide spectrum of biomolecules that are either synthesized in situ or transported from the blood capillaries into salivary acinar cells [2]. Not only can human saliva provide important information about the oral cavity, it also harbors a wealth of information that is derived from tissues/organs in the body. Such information can be used to assess and monitor health, well-being and disease status or alternatively be used to determine response to drug treatments [3], [4], [5], [6], [7].

A recent study by Bandhakavi et al. has identified 2340 proteins in human saliva and approximately 20% of these proteins are also found in plasma [8]. This highlights the importance and potential of saliva as a biological fluid and warrants more comprehensive evaluation as a diagnostic medium. There has been increasing interest in saliva diagnostics due to its non-invasive nature and ease of collection [3], [4], [5], [9], [10], [11], [12], [13]. Much of the literature on the use of saliva research focuses on developing technologies for the detection of biomolecules [5], [11], [12], [13], [14], [15]. However, important consideration must also be given to the effects of different saliva collection and processing methods and their influence on the accuracy of results as well as reference interval or cut off limits [8], [9], [10], [11], [12], [13], [15], [16].

Currently, saliva assays are routinely used to determine, disease such as HIV [17], illicit drug use [16], [17], cortisol levels for diagnosing Cushing's syndrome [18] and to measure many other hormones [19], [20], [21]. Saliva has numerous advantages over blood or urine as a diagnostic fluid: a) the non-invasive nature of sample collection and simple, safe, painless, and cost-effective; b) unskilled people can collect saliva samples at multiple time points; c) the total protein concentration is approximately a quarter of what is present in plasma which makes it easier to investigate low abundant proteins [5], [6], [22]. Human saliva can be collected either under resting conditions or stimulated conditions, such as mechanical stimulation via chewing onto an absorbent pad or acid stimulation [16], [23], [24]. Various methods of collecting human whole saliva samples, such as draining, spitting, suction and swabbing have been extensively reviewed [13], [23]. Currently, there are a number of commercially available saliva collection devices, namely Salivette® (Sarstedt, Germany), Salimetrics® Oral Swab (SOS) (USA), and Greiner Bio-One, Saliva Collection System (GBO SCS®) (Austria).

Biomarkers with clinical relevance [25] were selected and measured in the saliva collected from healthy participants in an attempt to provide baseline concentrations for these biomolecules in saliva. We selected CRP (115 kDa, inflammation marker) [25], [26], [27], IgE (160 kDa, allergy status biomarker) [28] and myoglobin (16.7 kDa muscle injury marker) [29].

The aims of our study were to evaluate the suitability of commercially available saliva collection devices (drool; SOS; Salivette® and SCS®) for protein detection using immunoassays and also to provide an indication of the levels of CRP, myoglobin, and IgE in the saliva of healthy individuals.

Section snippets

Participants

This study was approved by the University of Queensland Medical Ethical Institutional Board and all participants were required to sign an informed consent. The participant population consisted of 17 healthy volunteers: 7 women, mean age 25 years and 10 men, mean age 25 years. Exclusion criteria included existence of any co-morbid and/or oral disease (e.g. periodontal disease and gingivitis), autoimmune, infectious, musculoskeletal, or malignant disease, and recent operation or trauma, identified

Salivary flow rates in a healthy population

Unstimulated saliva collection methods namely, drool method and SOS, gave the lowest salivary flow rates 0.60 ± 0.39 and 0.47 ± 0.29 (mL/min) respectively. The mechanically stimulated salivary flow rates for the Cotton and Synthetic swabs resulted in significantly (p < 0.05) higher flow rates 1.10 ± 0.57 and 1.22 ± 0.46 (mL/min) when compared with the unstimulated saliva collection methods. Acid stimulated saliva collection method (SCS®) yielded the highest salivary flow rate 2.56 ± 0.95 (mL/min) (p < 0.05)

Discussion

In the present study, we investigated salivary stimulation methods and commercially available saliva collection devices for their suitability for the detection of proteins present in saliva in an emerging field of saliva diagnostics. We have determined preliminary reference intervals for the three anayltes measured in saliva for different collection devices and different stimulation methods within a healthy population. Although, the IgE median values suggest there may be difference between

Acknowledgements

The authors would like to acknowledge the financial support from the Queensland Government Smart Futures Fellowship Programme (QGSFF), the University of Queensland New Staff Research Funds (UQNSRSF 601252) and The University of Queensland Foundation Research Excellence Award. The authors wish to acknowledge Ms. Thea Cullen for her assistance in data collection. In addition, authors wish to acknowledge PerkinElmer for the supply of Beta-Test kits.

References (35)

  • C.K. Yeh et al.

    Current development of saliva/oral fluid-based diagnostics

    Tex Dent J

    (2010)
  • T. Pfaffe et al.

    Diagnostic potential of saliva: current state and future applications

    Clin Chem

    (2011)
  • Y.H. Lee et al.

    Saliva: an emerging biofluid for early detection of diseases

    Am J Dent

    (2009)
  • J.R. George et al.

    Future applications of oral fluid specimen technology

    Am J Med

    (1997)
  • S. Bandhakavi et al.

    A dynamic range compression and three-dimensional peptide fractionation analysis platform expands proteome coverage and the diagnostic potential of whole saliva

    J Proteome Res

    (2009)
  • M.E. Arellano-Garcia et al.

    Multiplexed immunobead-based assay for detection of oral cancer protein biomarkers in saliva

    Oral Dis

    (2008)
  • M. Choi

    Saliva diagnostics integrate dentistry into general and preventive health care

    Int J Prosthodont

    (2010)
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