Saliva Collection Methods Research Articles

Comparison of Saliva Collection Methods in Children with High-Functioning Autism Spectrum Disorders: Acceptability and Recovery of Cortisol

This study compared cortisol concentrations yielded using three saliva collection methods (passive drool, salivette, and sorbette) in both in vitro and in vivo conditions, as well as method acceptability for a sample of children (n = 39) with High Functioning Autism Spectrum Disorders. No cortisol concentration differences were observed between passive and sorbette samples obtained in vitro or in vivo. The salivette derived concentration was lower than the other two methods for the in vitro derived comparisons but did not differ from the other methods when collected in vivo. Cross-day comparison for the salivettes was also found to differ significantly, whereas the cross-day comparisons did not differ for the passive method or the sorbette method. Overall, passive drool and sorbettes were found to produce similar and stable readings of cortisol, whereas the salivette yielded unstable and variable concentrations. Ratings suggested that the children generally perceived all methods as acceptable.

Comparative Behavioral and Protein Study of Salivary Secretions in Homalodisca spp. Sharpshooters (Hemiptera: Cicadellidae: Cicadellinae)

Abstract A novel brush-induced method to physically stimulate salivation was applied to the glassy-winged and smoke tree sharpshooters (Hemiptera: Cicadellidae: Cicadellinae). This technique enabled the direct observation of salivary secretion processes, solidification of saliva, and collection of salivary secretions. For both species, brush-induced saliva was first secreted in liquid form, a portion of which gradually solidified to form the salivary sheath for both sharpshooter species. Proteins of similar molecular weight were obtained from brush-induced saliva extracts from both sharpshooters. Extracts from dried sheaths collected from Parafilm membranes over artificial diet had a different protein profile from brush-induced saliva extracts. The latter contained fewer proteins than extracts of the liquid content of salivary glands. Two proteins appeared in all three of the extracts from hemolymph, salivary glands, and brush-induced saliva, one of which also appeared in dried sheath extracts. Our findings support previous research by others that there is a limited flow of protein from hemolymph to salivary glands and brush-induced saliva. There is also some protein modification associated with saliva solidification. The quantity and composition of proteins suggest the brushinduced saliva collection method has merit for future biochemical analyses of saliva. The implications of this work could potentially include illuminating the mechanism of inoculation by the Pierce's disease bacterium, Xylella fastidiosa.

DNA yield and quality of saliva samples and suitability for large-scale epidemiological studies in children

To evaluate two saliva collection methods for DNA yield and quality as applied to a large, integrated, multicentre, European project involving the collection of biological material from children. Cross-sectional multicentre comparative study in young children. Saliva samples were collected from 14,019 children aged 2-9 years from eight European countries participating in the IDEFICS (Identification and prevention of dietary- and lifestyle-induced health effects in children and infants) study. This involved either the collection of 2 ml of saliva from children who were able to spit, or using a sponge to collect whole saliva and buccal mucosal cells from the inside of the mouth of younger children unable to spit. Samples were assembled centrally in each participating centre and subsequently despatched for DNA extraction and biobanking to the University of Glasgow. A subgroup of 4678 samples (∼33% of sampled individuals) were chosen for DNA extraction before genotyping. The whole-saliva collection method resulted in a higher DNA yield than the sponge collection method (mean±s.d.; saliva: 20.95±2.35 μg, sponge: 9.13±2.25 μg; P<0.001). DNA quality as measured by A (260)/A (280) was similar for the two collection methods. A minimum genotype calling success rate of 95% showed that both methods provide good-quality DNA for genotyping using TaqMan allelic discrimination assays. Our results showed higher DNA yield from the whole-saliva collection method compared with the assisted sponge collection. However, both collection methods provided DNA of sufficient quantity and quality for large-scale genetic epidemiological studies.

BRAIN-DERIVED NEUROTROPHIC FACTOR IN HUMAN SALIVA: ELISA OPTIMIZATION AND BIOLOGICAL CORRELATES

In order to assess the physiological significance of human salivary brain-derived neurotrophic factor (BDNF), we have optimized a sensitive and specific enzyme-linked immunosorbent assay (ELISA). We determined the range of salivary BDNF concentrations, the impact of saliva collection method, and the association of salivary BDNF with several biological characteristics. The ELISA had a detection limit of 62.5 pg/mL, and intra-assay and interassay precisions of 4.2% and 8.2%, respectively. Salivary BDNF concentrations were highly variable between individuals (median = 618 pg/mL) and were affected by collection method. Women had significantly higher levels of salivary BDNF than men. There was no relationship, however, between salivary BDNF levels and the other biological characteristics examined.

Measuring DHEA-S in saliva: time of day differences and positive correlations between two different types of collection methods

BackgroundThe anabolic steroid, dehydroepiandosterone sulfate (DHEA-S), is secreted from the adrenal cortex. It plays a significant role in the body as a precursor to sex steroids as well as a lesser known role in the hypothalamic pituitary adrenal axis (HPA) response to stress. DHEA-S can be measured reliably in saliva, making saliva collection a valuable tool for health research because it minimizes the need for invasive sampling procedures (e.g., blood draws). Typical saliva collection methods include the use of plain cotton swab collection devices (e.g., Salivette®) or passive drool. There has been some speculation that the plain saliva cotton collection device may interfere with determination of DHEA-S by enzyme immunoassay (EIA) bringing this saliva collection method into question. Because of the increasing popularity of salivary biomarker research, we sought to determine whether the cotton swab interferes with DHEA-S determination through EIA techniques.FindingsFifty-six healthy young adult men and women aged 18-30 years came to the lab in the morning (0800 hrs; 14 men, 14 women) or late afternoon (1600 hrs; 14 men, 14 women) and provided saliva samples via cotton Salivette and passive drool. Passive drool collection was taken first to minimize particle cross contamination from the cotton swab. Samples were assayed for DHEA-S in duplicate using a commercially available kit (DSL, Inc., Webster, TX). DHEA-S levels collected via Salivette and passive drool were positively correlated (r = + 0.83, p < 0.05). Mean DHEA-S levels were not significantly different between collection methods. Salivary DHEA-S levels were significantly higher in males than in females, regardless of saliva collection method (p < 0.05), and morning DHEA-S values were higher than evening levels (p < 0.05).ConclusionsResults suggest that DHEA-S can be measured accurately using passive drool or cotton Salivette collection methods. Results also suggest that DHEA-S levels change across the day and that future studies need to take this time of day difference into account when measuring DHEA-S.

Array-Based Whole-Genome Survey of Dog Saliva DNA Yields High Quality SNP Data

BackgroundGenome-wide association scans for genetic loci underlying both Mendelian and complex traits are increasingly common in canine genetics research. However, the demand for high-quality DNA for use on such platforms creates challenges for traditional blood sample ascertainment. Though the use of saliva as a means of collecting DNA is common in human studies, alternate means of DNA collection for canine research have instead been limited to buccal swabs, from which dog DNA is of insufficient quality and yield for use on most high-throughput array-based systems. We thus investigated an animal-based saliva collection method for ease of use and quality of DNA obtained and tested the performance of saliva-extracted canine DNA on genome-wide genotyping arrays.Methodology/Principal FindingsOverall, we found that saliva sample collection using this method was efficient. Extractions yielded high concentrations (∼125 ng/ul) of high-quality DNA that performed equally well as blood-extracted DNA on the Illumina Infinium canine genotyping platform, with average call rates >99%. Concordance rates between genotype calls of saliva- versus blood-extracted DNA samples from the same individual were also >99%. Additionally, in silico calling of copy number variants was successfully performed and verified by PCR.Conclusions/SignificanceOur findings validate the use of saliva-obtained samples for genome-wide association studies in canines, highlighting an alternative means of collecting samples in a convenient and non-invasive manner.

Imunoglobulina A salivar (IgA-s) e exercício: relevância do controle em atletas e implicações metodológicas

O objetivo do presente estudo é realizar uma análise e discussão dos principais achados envolvendo o comportamento da imunoglobulina A salivar (IgA-s) em relação ao estímulo do exercício e evidenciar a importância de seu controle em atletas. O exercício físico é um importante modulador das características do sistema imune, sobretudo do comportamento da IgA-s, componente fundamental na proteção de infecções do trato respiratório superior (ITRS). No entanto, a relação direta entre baixas concentrações de IgA-s e ITRS precisa de mais evidências para ser confirmada. As concentrações de IgA-s durante e logo após um exercício intenso diminuem, deixando o indivíduo mais suscetível à infecção, porém, atletas envolvidos em atividades extenuantes não são clinicamente imunodeficientes, comparados com indivíduos sedentários. Essas modificações são transitórias, com retorno aos valores normais após aproximadamente 48 horas de repouso. A razão dessas alterações não é clara, mas se apresenta multifatorial: elevação de hormônios estressores; fatores nutricionais; ação de espécies reativas de oxigênio; e estresse psicológico. Apesar do efeito transitório das alterações provocadas nos elementos do sistema imune frente ao exercício, observa-se diferença na variabilidade da IgA-s em populações com diferentes níveis de condicionamento. Diferenças metodológicas - como o protocolo de exercício, o método de coleta, armazenamento e manipulação da saliva, a forma de expressão da IgA-s, o nível de hidratação, o controle da dieta, a sazonalidade do período de competição, a aclimatação entre outros fatores - devem ser levadas em consideração para comparação entre os estudos. Além disso, ajudam a explicar, em parte, os resultados adversos envolvendo exercício moderado e os intermitentes, em que se encontram aumento, redução e até ausência de alteração nos níveis de IgA-s. Investigações de elementos inovadores, como os toll-like receptors, e o avanço tecnológico podem colaborar para aumentar as evidências sobre o tema.

Detection of oral squamous cell carcinoma using epigenetic biomarkers in saliva derived DNA

Introduction: Recent work on dysplastic lesions has shown that promoter methylation may be a predictive biomarker for malignant transformation to squamous cell carcinoma. Saliva shows promise as a tumour surrogate, but has not been investigated in the context of dysplasia. We investigated two methods of saliva collection for DNA yield and quality, as determined by epigenetic marker analysis, and the logistics of collection and storage.

Drooling in cerebral palsy: hypersalivation or dysfunctional oral motor control?

To investigate whether drooling in children with cerebral palsy (CP) in general and in CP subtypes is due to hypersalivation. Saliva was collected from 61 healthy children (30 males, mean age 9y 5mo [SD 11mo]; 31 females, mean age 9y 6mo [1y 2mo]) and 100 children with CP who drooled (57 males, mean age 9y 5mo [3y 11mo], range 3-19y; 43 females, mean age 10y 1mo [4y 9mo], range 4-19y), of whom 53 had spastic, 42 had dyskinetic, and five had ataxic CP. Almost all children were affected bilaterally, and 90 of them were at Gross Motor Function Classification System levels III or higher. The saliva was collected by the swab saliva collection method. The intensity of drooling was evaluated using the drooling quotient. No difference was found in the flow rates, age, or sex between healthy children and children with CP who drooled. On additional subgroup analysis, the flow rates of children with dyskinetic CP differed statistically from those of healthy children (submandibular p=0.047, parotid p=0.040). This study supports the finding in previous studies that no hypersalivation exists in children with CP who drool. Dysfunctional oral motor control seems to be responsible for saliva overflow from the mouth, whereas increased unstimulated salivary flow may occur in children with dyskinetic CP as a result of hyperkinetic oral movements.

A re-investigation of saliva collection procedures that highlights the risk of potential positive interference in cortisol immunoassay

Background The use of saliva for measurement of cortisol permits non-invasive study of adrenal function, but collection can be technically difficult, particularly in small infants. Saliva collection can be assisted by citric acid to increase saliva flow, or by the use of cotton or polyester swabs in the mouth. Aim To determine whether different methods of saliva collection affect cortisol radioimmunoassay (RIA) performance. Experimental Cortisol was measured in saliva collected from 16 adults using intra-oral cotton swabs or polyester swabs, compared with saliva dribbled directly into a pot either alone (plain saliva) or after citric acid had been placed on the tongue. An in-house RIA, without prior extraction, was used to measure cortisol with an encapsulated sheep antibody. Results Mean (median) salivary cortisol was 10.9 (10.5) nmol L −1 in plain saliva, 10.4 (8.4) nmol L −1 in citric acid stimulated saliva; 25.3 (25.1) nmol L −1 in saliva collected on cotton swabs, and 27.9 (27.3) nmol L −1 collected on polyester swabs. Cortisol in saliva collected using citric acid was not significantly different from plain saliva ( p = 0.997), but cortisol in saliva collected using cotton and polyester swabs was significantly higher than that of plain saliva ( p < 0.01). Conclusion The use of cotton or polyester swabs for collection of saliva can result in spuriously high levels of cortisol when measured by RIA.

Acute and chronic effects of exercise on markers of mucosal immunity

Decreased secretion rate of salivary markers of mucosal immunity, and in particular salivary immunoglobulin A (s-IgA), have been implicated as risk factors for subsequent episodes of respiratory infection in athletes. IgA is the predominant Ig in mucosal secretions and acts with innate mucosal defences to provide the 'first line of defence' against pathogens and antigens presented at the mucosa. As well as summarising the evidence concerning the effects of acute exercise and longer-term intensive training on these markers of mucosal immunity, this review explores the factors that impact upon salivary responses to exercise, such as method of saliva collection, stimulation of saliva collection and the method of reporting s-IgA data. The influence of adequate hydration and nutritional supplementation during exercise as well as exercising in extreme environmental conditions on salivary responses is also explored. Finally, the possible mechanisms underlying the acute and longer-term of effects of exercise on salivary responses are examined, with particular emphasis on the potential role of the sympathetic nervous system and the expression and mobilisation of the polymeric Ig receptor.

Evaluation of New Saliva Collection Device for Determination of Salivary Cotinine, Cortisol, Dehydroepiandrosterone and Testosterone Concentrations

This study was conducted to examine the effectiveness of a new salivary collection device (psi10 mm x 25 mm) made of polypropylene and polyethylene polymers (Salisoft((R))). Experiment 1; We measured the absorption capacity of the new device by two methods. Next, we examined whether the device affected the pH of 0.1 M NaHCO(3) solution (pH 8.7) and 0.1 M sodium phosphate buffer solution (pH 6.0). Experiment 2; We compared three saliva collection methods: by passive drool, using a cotton device (Salivette((R))), and using the new device. Saliva samples were collected from twelve men (average age, 31.5 +/- 17.1 years). Saliva samples were assayed for cotinine, cortisol, dehydroepiandrosterone, and testosterone concentrations by enzyme immunoassay, and the pH of saliva samples were measured. After this device was put in the mouth for one minute, 1.28 +/- 0.13 mL (mean +/- SD, N = 6) of saliva samples were obtained. The mean pHs of saliva samples collected using Salisoft((R)) and by passive drool did not differ significantly, whereas that of saliva samples collected with Salivette((R)) was significantly low. Saliva samples collected with Salisoft((R)) and those obtained by passive drool did not show significant differences in the concentrations of cotinine, cortisol, dehydroepiandrosterone, and testosterone. Moreover, significant positive correlations were noted between the concentrations in saliva samples collected with Salisoft((R)) and those in saliva samples collected by passive drool. This new device was shown to be suitable for saliva collection for the determination of the concentrations of cotinine and some steroids by enzyme immunoassay.

To spear or not to spear: Comparison of saliva collection methods

The eye spear, or an absorbent sponge-like material, has been proposed as a useful method of obtaining repeated saliva samples from infants and young children for cortisol determination. This brief report examines possible interference effects of different types of eye spears under conditions of relatively high and low cortisol levels, with or without the use of oral stimulant, and using two common assays. In Study 1, one type of eye spear was compared to passively collected drool using two different assays (EIA, DELFIA), across high and low concentrations of cortisol. No differences were found between methods for either assay or cortisol level, indicating that the spears are potentially a viable method of collecting saliva. Study 2 compared three other types of absorbent eye spears to passive drooling under the presence or absence of oral stimulant use. This study revealed that the degree of interference varied as a function of the specific type of eye spear that was employed; stimulant use had no effect. Taken together, the results raise important considerations to take into account when selecting collection materials and procedures in the measurement of salivary cortisol.

Measuring salivary flow: Challenges and opportunities

Saliva is being studied extensively and is being used for risk assessment, diagnosis and monitoring high-risk behavior and disease progression. A variety of medical conditions and medications are associated with salivary gland hypofunction. The major disadvantage in the use of saliva for health-related purposes is the lack of standardization in saliva collection methods. The authors provide a brief overview of different methods of saliva collection and the advantages and disadvantages associated with each method, as well as of how to assess the salivary flow rate. The authors present the complete set up and step-by-step guidelines for the collection of unstimulated and stimulated whole saliva. The life expectancy of people will continue to increase with advances in medicine and therapeutic modalities, and the prevalence of salivary gland hypofunction in the elderly population will increase owing to their longevity. The assessment of salivary gland hypo-function will need to be incorporated into everyday clinical practice. The saliva collection methods outlined in this article can be used by dentists to assess patients at risk of developing diseases and by scientists for scholarly activities.

Marshmallows used as saliva stimulant do not affect cortisol concentrations: Finally a palatable alternative for toddler saliva collection

Two studies were conducted to validate marshmallows as a saliva stimulant for use with toddlers. First, cortisol concentrations from 14 subjects (ages 6-46 years) were compared using three saliva collection methods: (1) plain cotton dental roll, (2) dental roll with one mini-marshmallow, and (3) expectorating into a collection tube using no cotton or stimulant. EIA was used for analyses. There were no significant differences among cortisol concentrations. Second, saliva collection compliance rate was compared for 21-month-olds (n = 51) using either flavored drink crystal- (compliance rate = 16.7%) or marshmallow-flavored (compliance rate = 60%) dental rolls for saliva collection (chi(2) (1) = 4.02, p = .045). These studies indicate that marshmallow is a viable option for saliva stimulation to determine toddler cortisol concentrations using EIA.

Influence of the sample collection method on salivary interleukin–6 levels in resting and post-exercise conditions

Previous studies demonstrated that no significant relationships exist between salivary and serum IL-6 in resting conditions and following exercise and that appropriate saliva collection procedures allow to avoid analytical drawbacks. This investigation aimed to: (a) compare the effects of two methods of saliva collection on IL-6 assay; (b) search for correlation between salivary and serum IL-6 in resting and post-exercise conditions; (c) evaluate the IL-6 response to isometric contractions. Seventeen sedentary subjects and fifteen athletes underwent one blood and two salivary draws: saliva was collected chewing on cotton salivettes and using a plastic straw (SA method and ST method, respectively). Afterwards, the athletes only completed a fatiguing isometric exercise of the knee extensors and blood and saliva were sampled after the exercise. In the entire group (n=32), ST method produced higher IL-6 levels than SA method and serum sampling. The exercise elicited significant responses of lactate, serum IL-6, salivary IL-6 (by ST method): salivary IL-6 values using the ST collection method were higher at each sampling point than with the SA method. The correlation analyses applied to both resting levels in the entire group and absolute changes above baseline in the athlete group showed that: (1) no significant relationships exist between serum and salivary IL-6 levels; (2) the greater the salivary IL-6 measurement, the higher the resultant inaccuracy of the SA method; (3) significant correlations exist between isometric force and mechanical fatigue during exercise and peaks of lactate and serum IL-6. These data provided demonstration of a cotton-interference effect for the results of salivary IL-6 assay and confirmed the lack of significant correlation between salivary and serum IL-6 in resting and post-exercise conditions.

Saliva collection method affects predictability of serum cortisol

Background Measurement of cortisol is of particular importance for early diagnosis of adrenocorticotropic dysfunction and therapeutic monitoring. Saliva samples clearly present an easily manageable, non-invasive and anytime practicable assessment method of glucocorticoid production. The aims of the present study were to compare salivary cortisol collection method “Salivette” versus “passive drool”, salivary cortisol concentrations with calculated free serum cortisol (FSC) and total serum cortisol (TSC) and the prediction of serum cortisol by salivary cortisol. Methods Saliva samples were collected by Sarstedt-Salivette® and by passive drooling into plastic tubes. Salivary cortisol, TSC measurements and calculation of FSC concentrations as well were carried out simultaneously in 10 healthy volunteers on two consecutive days at six definite points of time (08.00–23.00). Results Significant differences of salivary cortisol concentrations dependent on the saliva collection method ( F-test 6.45; p = 0.0317) and points of times were found ( F-test 41.19; p < 0.0001). Stronger overall correlation was observed in Salivette-salivary cortisol vs. TSC ( r = 0.813) and FSC ( r = 0.836) as compared to passive drooling. Specifically, more significant correlations were established in Salivettes vs. TSC (8/12 points of time, 66.7%) and FSC (8/12 points of time, 66.7%) than in passive drooling (TSC: 1/12 points of time, 8.3% and FSC: 2/12 points of time, 16.7%). Importantly, cortisol in Salivettes was a better predictor for FSC and TSC than passive drooling. Conclusions Our data indicate that salivary cortisol measurement with Salivettes is a reliable prediction method of total and calculated free serum cortisol levels. Moreover, it is a convenient method for saliva collection, handling and laboratory processing.

Assessing cortisol and dehydroepiandrosterone (DHEA) in saliva: effects of collection method

An increasing number of studies are utilizing saliva sampling as a method of assessing adrenal steroid secretion. Saliva samples have certain advantages over plasma, being non-invasive and easily collected. However, some methods of collection may compromise the accuracy of the assay, particularly those which employ aids to stimulate saliva production. We sought to compare the accuracy of cortisol and dehydroepiandrosterone (DHEA) measurement by examining the association between plasma levels, saliva and saliva collected using a citric acid-treated salivette device. Twenty six healthy male volunteers were recruited for the study. To increase the range of steroid levels in the samples collected, half the subjects were pre-treated with hydrocortisone (20mg, twice a day for 7 days) and half with placebo. Saliva samples were then collected from each subject using both a 'passive drool' method and a citric acid-treated salivette. A plasma sample was also collected. Cortisol and DHEA levels were measured by radioimmunoassay. For cortisol levels, both methods of saliva collection correlated highly with plasma levels and with each other (r 0.85; R(2) 0.72 for all). For DHEA levels, only saliva samples collected using the unstimulated collection method correlated with plasma levels. DHEA collected using the salivette device did not correlate significantly with either plasma or the unstimulated saliva (r 0.2;R(2) 0.04). It is crucial that future studies are aware of these issues and are cognizant of the effects of the method of collection when examining steroid levels in saliva.

Quality and Quantity of Saliva DNA Obtained from the Self-administrated Oragene Method—A Pilot Study on the Cohort of Swedish Men

Self-collection of saliva has the potential to provide molecular epidemiologic studies with DNA in a user-friendly way. We evaluated the new Oragene saliva collection method and requested saliva samples by mail from 611 men (ages 53-87 years). We obtained a response rate of, on average, 80% [varying from 89% (ages 67-71 years) to 71% (ages 77-87 years)]. DNA was extracted from 90 randomly selected samples, and its usefulness was evaluated with respect to quality, quantity, and whole-genome amplification (WGA). Visual inspection of DNA on agarose gels showed high molecular weight DNA (>23 kb) and no degradation. Total DNA yield measured with PicoGreen ranged from 1.2 to 169.7 mug, with a mean of 40.3 mug (SD, 36.5 mug) and a median of 29.4 mug. Human DNA yield was estimated by real-time PCR of the human prothrombin gene to account for 68% (SD, 20%) of total DNA. We did WGA on 81 saliva DNA samples by using the GenomiPhi DNA kit and genotyped both saliva DNA and WGA DNA for 10 single-nucleotide polymorphisms randomly selected from the human genome. Overall genotyping success rate was 96% for saliva DNA and 95% for WGA DNA; 79% of saliva DNA samples and 79% of WGA DNA samples were successfully genotyped for all 10 single-nucleotide polymorphisms. For the 10 specific assays, the success rates ranged between 88% and 100%. Almost complete genotypic concordance (99.7%) was observed between saliva DNA and WGA DNA. In conclusion, Oragene saliva DNA in this study collected from men is of high quality and can be used as an alternative to blood DNA in molecular epidemiologic studies.

Salivary cortisol in preterm infants: Validation of a simple method for collecting saliva for cortisol determination

Background and aims The increased use of salivary cortisol as a biomarker of stress and/or diurnal rhythms has facilitated research of Hypothalamic–pituitary–adrenocortical (HPA) function. Saliva collection remains problematic with preterm infants. The twofold purpose of this study is to 1) establish validity of the filter paper method for saliva collection and 2) apply the filter paper method for saliva collection to preterm infants. Design and measures Whole saliva was collected from six normal adult subjects to create a pool. Validation measures included comparison of levels obtained from whole saliva and filter paper, an evaluation of storage effects, assessing spiking recovery, and measurement of linearity of dilution. In the application study, saliva was collected every three hours, before feedings for three consecutive days from 26 hospitalized preterm infants. Diurnal variation in cortisol was examined using hierarchical linear modeling and individual calculation of diurnal pattern using an accepted technique. Results Validation studies revealed acceptable recovery of whole saliva from filters, no effect of room temperature storage of filters for up to six months, and acceptable linearity of dilution up to 4×. Saliva from preterm infants was easily collected. Only 2% of the samples were lost due to inadequate wetting of the filters. An inverse association was found between postconceptional age and one-minute APGAR scores and infant cortisol levels. Variable daily cortisol patterns and no discernable rhythm were found for this sample; however, four infants appeared to show a typical diurnal pattern. Conclusions The filter paper method is a valid method of saliva collection that is feasible to use with preterm infants.