Table 2 Different types of saliva samples, times of the day for sampling, collection techniques, collection devices, patients’ preparations, sampling duration, transporting conditions, and restoring conditions

Lenander-Lumikari et al., 1995 [63]

1) WMS (stimulated)

2) At 9 AM and 11 AM

1) 16 (Healthy)

2) 5M:11F

3) Age range: 27 – 41

- All patients were instructed to eat breakfast between 7:30 and 8 AM

- No eating or smoking 1 h before sampling

- Total saliva quantity

- Salivary pH and buffering pH

- Salivary levels of calcium, sodium, potassium, phosphate, chloride and thiocyanate

- 3 methods were assessed:

A) Salivette® collection kits;

with a neutral, non-covered cotton roll (chewing for 3 min) (stimulated)

B) Salivette® collection kits;

with a polyether roll covered with polypropylene (chewing for 3 min) (stimulated)

C) Paraffin wax (melting point 46 – 48 °C); 1 g piece (control) (chewing) (stimulated), patients chewed on paraffin to softness, swallowed the produced saliva and then expectorated the secreted saliva into ice-chilled tubes for 3 min

- Patients were categorized into 2 groups (8 participants each); first group used technique A at both 9 and 11 AM in the same day and both times used technique C as control immediately after A. On a separate day, they used technique B first at 9 AM and C as control immediately after B

Second group had the same arrangement as group one but they used C as control before using A or B

- Salivette®: after chewing, rolls were placed in pre-weighed tubes, centrifuged at 500 g for 10 min at room temperature and then immediately pipetted into analytical tubes and frozen at − 20 °C

- Paraffin wax: expired air was blown over samples, tubes were sealed with M laboratory Parafilm®. For lysozyme, albumin and lactoferrin, saliva was kept uncentrifuged. For the remaining analyses, saliva was centrifuged at 14,500 g at 4 °C for 15 min and then immediately pipetted into analytical tubes and frozen at − 20 °C

- Total average volume of WMS:

1) Salivette® (non-covered): 2.4 mL

2) Salivette® (polypropylene-covered): 1.6 mL

3) Paraffin (control): 5.4 mL

- Results of pH, sodium, potassium, chloride and phosphate were similar in all 3 methods

- The buffering pH was significantly lower than control in both Salivette® techniques

- Calcium levels were significantly higher in Salivette® (non-covered), compared to the other 2 methods

- Thiocyanate levels were significantly higher in both Salivette® techniques

Ng et al., 2004 [65]

1) WMS (unstimulated)

2) NM

1) 10 (Healthy)

2) NM

3) NM

- No eating, drinking or smoking 1 h before sampling

- Salivary DNA purity

- Each patient provided 12 mL of unstimulated sample via passive drooling

- 12 mL unstimulated samples were mixed by vortexing and inversions, then five 2 mL aliquots were dispended into 15 mL tubes and were submitted to different storage procedures:

1) S1; washing samples with PBS and extraction on the same day of sampling

2) S2; washing samples with PBS then centrifuging to produce pellet. Storing the pellet at − 70 °C for 1 week before DNA extraction

3) S3; storing samples for 1 week at 4 °C, followed by washing samples and DNA extraction

4) S4; storing samples for 1 week at 4 °C, followed by washing samples and producing pellet, then storing the pellet for 1 month at − 70 °C before DNA extraction

5) S5; storing samples for 1 month at − 70 °C, followed by washing and DNA extraction

- Purity of DNA was similar in all 5 methods (based on OD_260/280 ratios)

- All 5 methods resulted in the presence of correct sized (581 bp) single specific product

- PCR bands quantification:

S1 > S2 > S3 > > S4 ≅ S5

Anthonappa et al., 2012 [60]

1) WMS (unstimulated)

2) NM

1) 50 (Healthy)

2) NM

3) Age range: 5 – 10

NM

- Salivary DNA quantity

- Salivary DNA purity

Each patient gave 2 mL of their WMS samples using a Oragene® self-collection DNA kit, in a single time period (spitting). The lid of the kit contains Oragene® DNA solution. After completion of sampling, WMS is immediately mixed with the solution, which stabilizes DNA and prevents bacterial growth

- 5 different restorage methods were used:

1) SC1; DNA extraction immediately after sampling

2) SC2; storing for 1 month at 37 °C before DNA extraction

3) SC3; storing for 6 months at 37 °C before DNA extraction

4) SC4; storing for 12 months at 37 °C before DNA extraction

5) SC5; storing for 18 months at 37 °C before DNA extraction

- DNA yield:

SC2 ≅ SC3 ≅ SC4 ≅ SC5 > > SC1

- DNA purity (based on both OD_{260/280 and} OD_260/230):

NSD

Durdiaková et al., 2013 [67]

1) WMS (unstimulated and stimulated)

2) Between 8 to 10 AM

1) 10 (Healthy)

2) 5M:5F

3) Age range: 19 – 21

- No eating, drinking or oral hygiene procedures 30 min before sampling

- No coughing during sampling (in order to avoid mucus entering the sample)

- Salivary testosterone

- All patients were asked to take all following samples with 5 min intervals;

- Control; Patients dropped down their heads and let their WMS run naturally and spit it out after a while (2 mL). Control samples were not centrifuged

- Repeatedly collected saliva (RS); for analyzing the effects of centrifugation immediately after sampling (the clear top-phase (100 μL) was used for ELISA):

1) RS1: centrifuged at 2,000 g for 5 min (1 mL)

2) RS2: centrifuged at 6,000 g for 5 min (1 mL)

3) RS3: centrifuged at 10,000 g for 5 min (1 mL)

- Stimulated saliva (2 mL); patients were asked to touch the tip of their tongues several times with a coated cotton swab (soaked in 2% citric acid). SS samples were not centrifuged

- Following methods were used to test the salivary testosterone levels:

A) Comparing the testosterone levels of unstimulated (control) and stimulated without centrifugation

B) Analyzing the effect of centrifugation; comparing the results of centrifuged unstimulated samples (RS1, RS2 and RS3) against control. All of the samples were fresh and were not frozen for their ELISA assays. No processing was performed

C) Analyzing the effect of different restoration temperatures and restoration times; unstimulated samples were stored in different conditions (room temperature, 4 °C, − 20 °C and − 80 °C) immediately after sampling. Samples were stored for 1 day, 1 week or 1 month. On the day of analysis, restored samples were brought to room temperature and freshly collected unstimulated was used as control

- Comparing the testosterone levels of unstimulated (control) and stimulated without centrifugation:

NSD between control and stimulated

- B) Analyzing the effect of centrifugation: the testosterone levels were significantly higher in control:

Control > > RS1 ≅ RS2 ≅ RS3

- C) Analyzing the effect of different restoration temperatures and restoration times:

Testosterone levels were not significantly different in different restoration periods (1 day, 1 week, or 1 month) in each of the restoration conditions (room temperature, 4 °C, − 20 °C and − 80 °C)

Peres et al., 2015 [51]

1) WMS (unstimulated and stimulated)

2) A) Basal and lozenge day: 9:45 AM, 10:00 AM, 10:15 AM, 10:30 AM and 10:45. AM

B) Bacon day: 9:45 AM and 10:00 AM

1) 47 (Healthy)

2) 22M:25F

3) Age range: 20 – 34

- No eating or drinking 1 h before sampling

- No use of antihistamines, antihypertensives, anticholinergics or diuretics

- Patients with dislike of bacon or self-reported vegetarianism were excluded

- Patients mouth-rinsed with water 5 min before sampling

- Salivary cortisol,

- Salivary DHEA

- Salivary testosterone

- The following 3 methods were used to collect patients’ saliva (2 mL from each method through a short plastic straw into a collection vial)

A) An OTC anhydrous crystalline dietary supplement in lozenge form (Maxisal™) was analyzed for its saliva increasing abilities (lozenge day). One lozenge was administrated by each patient 25 min before sampling (stimulated)

B) The smell of freshly baked bacon (microwaved 5 min before patients’ arrival, and left in front of patients for 5 min before sampling) was used to increase patients’ saliva flow (bacon day) (stimulated)

C) Regular non-stimulated passive drooling saliva sampling (basal day) (control) (unstimulated)

- For methods A and C patients provided 5 samples (2 mL each) with 15-min intervals. While for bacon method, only 2 samples were provided

- All samples were immediately frozen and stored at − 80 °C

- On the day of analysis, samples were thawed and centrifuged at 1,500 g for 15 min

- Sampling duration:

Lozenge > Bacon > > control

- Cortisol, DHEA and testosterone concentrations: NSD amongst the 3 methods. However concentrations of all 3 hormones decreased throughout morning sessions in all 3 methods

Justino et al., 2017 [55]

1) WMS (unstimulated and stimulated)

2) Between 8 and 9 AM

1) 14 (Healthy)

2) 7M:7F

3) Mean age: 21 ± 2

- No eating, drinking or smoking 2 h and 30 min before sampling

- Patients had to mouth-rinse with distilled water 5 min before sampling

- The first 2 min of saliva sampling was discarded

- Salivary flow rate

- Salivary total protein

- Salivary nitrite

-Salivary alpha amylase

-Salivary antioxidant capacity

All 6 following US and SS samples were collected for all participants at the same daytime, with 5 min intervals;

- Unstimulated

1) Patients accumulated saliva in their mouths for 30 s, then spat it into a tube, then 90 s of continuous spitting (US1)

2) Patients accumulated saliva in their mouths for 60 s, then spat it into a tube (US2)

3) 90 s of continuous spitting without accumulation (US3)

- Stimulated:

1) Patients circulated the Salivette® cotton inside their mouth for 90 s, then the cotton was placed inside a plastic tube (SS1)

2) Patients chewed Parafilm® for 30 s, spat the accumulated saliva into tubes, followed by 90 s of continuous spitting (SS2)

3) Patients chewed mint flavored gum for 30 s, spat the accumulated saliva into tubes, followed by 90 s of continuous spitting (SS3)

All samples were immediately stored at 4 °C until all 6 sampling steps were performed for each patient, then they were centrifuged at 1976 g at 4 °C for 15 min. The supernatants were analyzed and samples were stored at − 80 °C

- Mean salivary flow rate:

SS3 (3.42) > > SS1 (1.57) ≅ SS2 (1.56) ≅ US3 (1.31) > US2 (0.91) ≅ US1 (0.82)

- Total protein:

SS3 > > SS2 > US3 > SS1 ≅ US2 > US1

- Nitrite:

SS3 > > SS2 ≅ SS1 ≅ US3 ≅ US1 > US2

- Alpha amylase:

SS3 > SS1 ≅ SS2 ≅ US1 > US2 > US3

- Total antioxidant capacity:

SS3 > > SS1 ≅ SS2 ≅ US1 ≅ US2 ≅ US3

Karched et al., 2017 [64]

1) WMS (stimulated)

2) NM

1) 4 (Healthy)

2) NM

3) Age range: 34 – 41

NM

- Salivary DNA quantity

- Salivary bacterial quantity

Patients chewed paraffin wax and 4 mL of stimulated samples were collected

Samples (4 mL) were divided in 3 equal 1.3 mL aliquots. One tube was left on ice (WS), while the other 2 got centrifuged at 14,000 g at 4 °C for 15 min. Then the supernatant in one tube and pellet in the other tube were separated for analysis

All 3 tubes were subjected to DNA purification (Masterpure™ DNA purification kit) and were preserved at − 20 °C. Samples were tested at 0 days, 7 days, 2 months, and 6 months of restoration for DNA concentration and bacterial quantities (PCR)

- DNA concentration (at all 4 time points):

1) WS ≅ pellet > > supernatant

2) There were no significant differences in different evaluation periods in each group

- Mean bacterial quantities (cells/mL):

1) WS ≅ pellet > > supernatant

2) F. Nucleatum cell numbers in both WS and pellet, were significantly higher in 2 and 6 months restored samples, compared to 0 and 7 days

3) F. Alocis cell numbers in both WS and pellet, were significantly lower in 2 months of restoration compared to other time points

Lim et al., 2017 [66]

1) WMS (unstimulated)2) NM

1) 40 (Healthy)

2) NM

3) Age range: 20 – 30

- No eating or drinking 1 h before sampling

- No history of drinking and drinking habits

- No use of local and/or systemic antibiotics

- Patients mouth-rinsed with bottled water before sampling

- Salivary DNA quantity

- Salivary DNA quality

- Salivary bacterial gDNA purity

- 10 of the patients (Group 1) were asked to collect their saliva using both following methods with 5-min intervals:

A) Spitting into a 50 mL sterile Falcon tube

B) Spitting (1 mL) into a OMNIgene™ tube (2 mL); containing 1 mL of stabilizing buffer

- The remaining 30 patients (Group 2) were asked to collect their samples using all 3 following methods with 5-min intervals:

A) Spitting into a 50 mL sterile Falcon tube

B) Passive drooling into a 50 mL sterile Falcon tube

C) Patients were asked to swish and gargle with saline solution (10 mL, 0.9% (w/v)) for 1 min before passive drooling into a 50 mL sterile Falcon tube

- Samples collected in Falcon tubes were mixed with PBS (1:1) before restoring and analysis

- All of the spit and drool samples were evenly aliquoted into 1.5 mL Eppendorf tubes and stored at − 80 °C

- Saline solution samples were centrifuged at 1,000 g at 4 °C for 15 min to separate cellular pellet. Then pellets were resuspended in 1 × PBS and aliquoted into 1.5 mL Eppendorf tubes and stored at − 80 °C

- Variant gDNA extraction kits were assessed for all samples; Maxwell® 16 LEV blood DNA kit, in-house phenol–chloroform and QIAamp DNA Microbiome kit

1) Group 1:

- NSD in quantity and quality of extracted gDNA amongst the 2 methods and NSD amongst the 3 different gDNA extraction kits

2) Group 2:

- Maxwell® resulted in highest quantities of extracted gDNA. However, the data were less variable when QIAamp was used

- Maxwell® showed purest qualities of gDNA (260/280 ratio)

3) Maxwell® provided the most enriched bacterial gDNA extraction (for the spit samples (50 mL Falcon tubes))

4) there were NSD between spit, drool and saline solution regarding purity of bacterial gDNA

Roth et al., 2017 [68]

1) WMS (unstimulated)

2) 30 min after awakening, 10 min before blood draw and 20 – 30 min after the blood draw

1) 11,390 (Healthy)

2) NM

3) Age range: 3 – 4.5 month infants

- Infants with high risk genetic markers for T1D

- No eating, drinking, crying or brushing teeth 30 min before sampling

- No use of oral steroids 30 days prior to sampling

- Salivary cortisol

- 3 samples were collected from each patient using Salimetrics® salivary collection kit:

1) In-home sampling by parents 30 min after awakening

2) In-lab sampling by study staff 10 min before a blood draw

3) In-lab sampling by study staff 20 – 30 min after a blood draw

- Each kit had 3 sorbettes (cotton pads on a stick) and a storage tube (9 sorbettes in total). All 3 sorbettes were placed under patients’ tongues (one after the other) until each cotton was saturated with saliva

- All 3 sorbettes from each sample were put in 1 storage tube. The storage tubes were centrifuges at 1,500 g for 15 min and sorbettes were discarded afterwards. Then samples were stored at − 70 °C

- Only 1.6% of samples were excluded due to insufficient quantities

All samples from 3 different methods had sufficient rates of cortisol for further analysis

Garbieri et al., 2017 [56]

1) WMS (unstimulated)

2) NM

1) 20 (Healthy)

2) NM

3) Age range: ≥ 18

- No eating, drinking, kissing or smoking 30 min before sampling

- Salivary DNA quantity

- Salivary DNA quality

- Passive drooling of 20 mL of US into a 50 mL polyethylene tube

- Samples were aliquoted into 1.5 mL microcentrifuge tubes

- Aliquoted samples were tested immediately after sampling (T0) or were stored at − 20 °C for 3 months (T3), 6 months (T6) and 12 months (T12) until analysis

- 5 different protocols were used for DNA extraction:

1) Manual purification of DNA using Oragene® DNA kit; 1 mL of saliva sample and 1 mL of suspension buffer (1:1)

2) QIAamp® DNA mini kit; with no suspension buffer

3) Samples were centrifuged for 5 min at 10,000 g, the supernatant was discarded and the pellet was resuspended in 1 mL of extraction buffer. Then 5 μL of proteinase K was added and tubes were vortexed and incubated overnight at a 56 °C water bath, samples were centrifuged again, 500 μL of 10 M ammonium was added and mixture was mixed manually for 3 to 5 min and followed by centrifuging for 15 min at 21,000 g at room temperature. Then 500 μL of its supernatant was mixed with 540 μL of cold isopropyl alcohol and were placed in refrigerator for 2 h and centrifuged for 20 min at 10,000 g at room temperature. The supernatant was discarded and 1 mL of 70% ethanol was added and tubes were centrifuged for 5 min at 10,000 g. The supernatant was discarded again and tubes were left open for 4 – 5 h. Finally DNA was hydrated in 50 μL of autoclaved deionizer water

4) InstaGene™ Matrix; 1.5 mL samples were centrifuged at 10,000 g at 4 °C for 5 min. Supernatant was discarded, 1 mL of physiological saline was added to solve the pellet, vortexed for 30 s, then centrifuged at 10,000 g at 4 °C for 5 min. 200 μL of InstaGene™ Matrix was added, vortexed for 30 s, incubated for 30 min at 56 °C, vortexed for 10 s, boiled at 100 °C for 10 min, vortexed for 10 s and finally centrifuged at 15,000 g at 4 °C for 5 min

5) InstaGene™ Matrix; similar to protocol 4 with the addition of Proteinase K and 1% SDS

1) DNA quantity:

- T0: total amount of extracted DNA; protocols 4 and 5 > > protocols 1, 2 and 3

- T3: NSD between protocols 1, 3 and 5 in DNA levels

- T6 and T12: NSD between protocols 1 and 4 in DNA levels

- T12: protocol 5 had significantly higher levels of extracted DNA

- Protocol 1: extracted DNA was efficient at all time points and the amount of DNA had NSD amongst the 3 different time points

- Protocol 2: always had significantly lower amounts of DNA compared to protocol 1 at all time points

- The storage time affected the DNA concentration only in protocol 3

- DNA concentration in protocol 3: T0 > > T3 > T6 > > T12

- The least amount of extracted DNA amongst all protocols and across all time points: protocol 3 at T12

2) DNA purity:

- DNA purity for each protocol (protocols 1, 2, 3 and 4) were similar in different time points (T0, T3, T6 and T12). While, DNA purity in protocol 5 was rarely within the purity limits

- At all time points, protocols 1 and 2 had the highest number of samples within the DNA purity limit

- Number of samples within the DNA purity limit in protocol 4: T0 > T3 > T6 > T12

3) Unfragmented DNA:

- T0, T3, T6 and T12: protocol 1 had 100% unfragmented DNA, which was significantly higher than protocols 2 (5%), 3 (0%), 4 (10%) and 5 (20%)

Portilho et al., 2017 [57]

1) WMS (unstimulated)

2) NM

1) 74 (32 HBV-infected and 42 healthy patients)

2) 25M:49F; 20M:12F HBV-infected and 5M:37F healthy patients

3) Mean age: 37.76 ± 11.89

- No eating or drinking, 1 h before sampling

- Salivary HBV DNA quantity

- 4 different methods were assessed from each patient on the same day:

1) Spontaneous spitting (1 – 2 mL)

2) Salivette®; the absorbent pad was placed inside the mouth for 2 min

3) Whatman FTA™ Cards; the foam tipper applicator was rubbed inside the cheek for 30 s, the applicator was pressed onto an FTA™ card until complete saturation

4) DNA-SAL™; applicator was rubbed inside the cheek for few seconds, then a small quantity of mouth rinse was swished and spat into the tube (along with the applicator)

1) Salivette®; 1 mL of PBS (pH 7.2) was added, then centrifuged at 2,000 g for 10 min

2) Whatman FTA™; cards were dried at room temperature for 1 h

HBV DNA was detected in all 4 methods. However, Salivette® had the best results

Scherer et al., 2017 [58]

1) WMS (unstimulated)

2) NM

1) 110 (Cocaine or crack-cocaine using patients)

2) 104M:6F

3) Mean age: 33.7 ± 9.4

NM

- Cocaine and crack presence in the saliva

- 2 methods were assessed for all patients:

1) MDML™ (1 mL); red lines mean positive results of drug abuse (10 ng/mL and 20 ng/mL detection cutoff). The device also stores a little bit of saliva

2) DDS2™ mobile system (0.6 mL); the collector swab is swabbed around the tongue, gums and inside cheeks (10 ng/mL and 30 ng/mL detection cutoff)

- DDS2™ was assessed immediately after MDML™ with no interval

- Samples were stored at − 80 ± 2 °C

- Results of the 2 tested devices/methods were compared to Liquid Chromatography-Mass Spectrometry (LC–MS)

- In comparison with LC–MS:

1) MDML™ (20 ng/mL cutoff):

-Sensitivity: 100%

- Specificity: 65.6%

- Accuracy: 70.9%

2) MDML™ (10 ng/mL cutoff):

-Sensitivity: 92.6%

- Specificity: 71.1%

- Accuracy: 76.6%

3) DDS2™ (30 ng/mL cutoff):

-Sensitivity: 100%

- Specificity: 77.77%

- Accuracy: 80%

4) DDS2™ (10 ng/mL cutoff):

-Sensitivity: 88.89%

- Specificity: 89.15%

- Accuracy: 89.09%

Ishikawa et al., 2017 [69]

1) WMS (unstimulated)

2) Between 8 AM and 12 PM (for hospitalized patients: 1.5 h (Group 1) and 3.5 h (Group 2) after breakfast and 12 h after dinner (Group 3))

1) 66 (22 oral cancer patients and 44 healthy controls)

2) 28M:38F

3) Age range: 21 – 94

- Patients mouth-rinsed with water immediately prior to sampling

- For controls: no eating or drinking, 1.5 h before sampling

- For all patients: no tooth-paste or rinse 1 h before sampling

- Oral cancer metabolites presence in the saliva

- Sampling for patients at home:

Passive drooling (400 μL); in 50 cc Falcon tubes over 5 – 10 min

- Sampling for hospitalized patients:

- Samples were immediately stored at − 80 °C until analysis

- Samples were thawed and centrifuged at 9,100 g for 2.5 h, at 4 °C through a 5-kDa cutoff filter

- Capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) was assessed to quantify charged hydrophilic metabolites

- Group 3 had significantly better results than Groups 1 and 2; indicating that a 12 h fasting before sampling is ideal for quantifying charged metabolites in oral cancer patients

- The salivary levels of 51 metabolites significantly differed in controls versus oral cancer patients

Cohier et al., 2017 [70]

1) WMS (unstimulated)

2) NM

1) 5 (Healthy)

2) NM

3) Age range: NM

NM

- Illicit drugs presence in the saliva

- 2 devices were assessed to identify the recent usage of illicit drugs:

1) Quantisal® (1 mL saliva and 3 mL preservation buffer); with an absorptive cellulose pad placed under the tongue until the indicator was completely blue

2) Certus® (1 mL saliva and 3 mL preservation buffer); with an absorptive polyethylene pad actively swabbed around tongue, gums and inside the cheeks until the indicator was completely blue

- Samples were stored at − 20 °C, 4 °C or room temperature for 1, 7 or 14 days

- On average the sampling duration for Quantisal® and Certus® were 3 min and 1 min respectively

- At 4 °C: all drugs were stable at all time points with both devices, except for codeine, buprenorphine and methamphetamine (which their concentrations decreased at day 14)

- At − 20 °C: concentrations of opiates and amphetamines decreased over the storage time with both devices

- Methadone was stable and detectable at all time points and storing temperatures except for day 7

Ambers et al., 2018 [54]

1) WMS (unstimulated)

2) NM

1) 4 (Healthy)

2) NM

3) NM

NM

- Salivary flow rate

- Salivary DNA quantity

MicroFLOQ® Direct swabs using the “MicroFLOQ® wet or dry traces collection procedure”

- samples were collected and diluted into 10%, 5% and 1%. Then 10 μL of all samples were pipetted onto glass microscope slides and left to dry overnight

- DNA extraction was performed using the Nucleic Acid Optimizer (NAO®) Baskets and the QIAamp® kit

- MicroFLOQ® Direct swabs: the wet method had significantly better outcomes

Rosenbaum et al., 2018 [50]

1) WMS (unstimulated)

2) Prior to bed (pre-bed samples) and immediately after waking up (waking samples)

- 2005:

1) NM (Healthy)

2) NM

3) Age range: 20.9 – 22.1

- 2014:

-

1) NM (Healthy)

2) NM

3) Age range: 29.6 – 31.3

NM

- Salivary cortisol

- Salivary secretory immunoglobulin A (sIgA)

1) 2005: patients used 1.8 mL vials. Patients were instructed to fill about 1.5 mL of passive drooling unstimulated (all 3 samples: pre-bed, waking and 30 min after waking samples; 1 of each, 3 samples in total). Samples were kept at room temperature until they were retrieved by an interviewer in the morning

2) 2014: patients used 4.0 mL vials. Patients were instructed to fill about 3 mL of passive drooling unstimulated and repeated the sampling 1 week apart (both pre-bed and waking samples; 2 of each, 4 samples in total). Samples were kept at room temperature until they were retrieved by an interviewer in the morning

Upon the arrival of tubes in the USA, all tubes were immediately stored at − 80 °C. On the day of analysis samples were centrifuged then had their supernatants separated aliquoted into smaller tubes

- Mean sampling times:

1) 2005; 10:41 PM (pre-bed) and 6:48 AM (waking)

2) 2014; 10:24 PM (pre-bed) and 7:02 AM (waking)

- Cortisol levels: samples that spent more time in the − 35 °C cooler, had significantly lower cortisol values

- sIgA: samples that spent more time in the − 35 °C cooler, had significantly higher sIgA values

Mandrell et al., 2018 [52]

1) WMS (unstimulated)

2) 3 h, 2 h and 1 h before bedtime, at bedtime and 1 h after bedtime

1) 64; 39 children and 25 adolescents (Healthy)

2) 32M:32F; 18M:21F (children) and 14M:11F (adolescents)

3) Age range: 7 – 20

NM

- Salivary flow rate

- Overnight in-home salivary melatonin (DLMO) collection via passive drooling

- Each patient had to collect five 100 μL samples

- Patients’ put their samples into a freezer immediately after sampling

- In the morning sample tubes were returned and immediately frozen and stored at − 80 °C

- NSD in salivary properties amongst all patients in both age groups

Fakhry et al., 2018 [53]

1) WMS (unstimulated) and oral secretions

2) NM

1) 90 (with intact uterus) (Healthy)

2) 0M:90F

3) Age range: 25 – 45

NM

- Salivary immune markers

- WMS: NM

- Oral secretions: a Merocel® ophthalmic sponge was placed under the tongue for 30 s then placed into a 5 mL screw cap cryovial

- Samples were first stored at 4 °C for 8 h and then at − 80 °C until the day of protein extraction

- On the day of analysis, samples were thawed at room temperature for 10 min, placed in a microcentrifuge with a 0.2 μm filter, then mixed with 300 μL of extraction buffer (10 mg/mL aprotinin in PBS with 10% sodium azide). The mixture was incubated for 30 min at 4 °C, then centrifuged for 30 min at 4 °C at 14,000 rpm. Finally samples were stored at − 20 °C. Concentration of immune markers were tested by Luminex multiplex assay

- Mean concentrations of 30 out of 37 tested immune markers were significantly higher in oral secretion samples compared to WMS

- Mean concentrations of IL-9, IL-33, IL-6, IL-13, TNF-α, GCSF and SCD401 were similar between the 2 methods

- Oral secretions had a significantly more variable range of immune markers compared to WMS

Dos Santos et al., 2018 [59]

1) WMS (unstimulated)

2) Between 8 and 10 AM

1) 26 (Healthy)

2) 14M:12F

3) Age range: 18 – 36

- No eating, drinking or brushing teeth 1 h before sampling

- Patients mouth-rinsed with water 10 min prior to sampling

- Salivary flow rate

- Salivary buffering capacity

- Salivary pH

- Salivary total protein

- Salivary enzymes activity

Passive drooling (5 mL) while patients were seated upright

- Samples were centrifuged at 10,000 g at 4 °C for 10 min

- A total of 9 aliquots were made:

1) 1 aliquot was analyzed immediately after sampling without freezing

2) 4 aliquots were stored at − 20 °C

3) 4 aliquots were stored at − 80 °C

- Frozen samples were stored for 3, 7, 14 and 28 days

- Salivary flow rate, buffering capacity, pH and total protein concentrations:

There were NSD amongst all samples (fresh or frozen) at all time points

- The activities of all enzyme were decreased in the supernatant overtime in both − 20 °C and − 80 °C stored samples. However, the activity decrease was significantly higher in − 20 °C samples

- At − 20 °C:

The activity of none of the enzymes were enough for analysis

- At − 80 °C:

Enzymatic analysis of ALT, ALP and LDH up to 3 days of storage were possible and up to 10 days of storage for TRAP and ACP

Novak et al., 2021 [72]

1) WMS (unstimulated)

2) NM

1) 52; 22 infants (under the age of 1 year) and 30 children (1 – 6 year old) (Healthy)

2) 28M:24F

3) Age range: 2 – 30 months,

MA: 23 months

- No oral and maxillofacial deformities

- Total saliva quantity

- 2 methods were assessed for each patient with a 5-min interval:

1) Oral swab using Salimetrics® SalivaBio’s Children’s Swab (SCS); placed inside the mouth for 2 min

2) Salivac®; pacifier-based collection device placed inside the mouth for 2 min

NM

- Mean average amount of collected saliva:

NSD; Salivac® (174 μL) > SalivaBio (158 μL)

Guo et al., 2021 [62]

1) WMS (unstimulated)

2) A) 6:00—6:30 AM; before breakfast

B) 9:00 – 9:30 AM; after breakfast

C) 11:00 – 11:30 AM; before lunch

D) 14:00 – 14:30 PM; after lunch

E) 16:30 – 17:00 PM; before dinner

F) 19:00 – 19:30 PM; after dinner

1) 29 (Healthy)

2) 20M:9F

3) Mean age: 10.17 ± 1.37

- No history of thyroid diseases and intake of iodine supplements

- Salivary iodine

Passive drooling using 15 mL screw polyethylene bottles; 2 ml of US samples for each of the 6 time points

- Samples were stored at room and tested 1 week after sampling

- 1 week after sampling; samples were centrifuged at 3,00 r/min for 5 min. 50 μL of saliva supernatant was mixed well with 0.95 mL 7 mmol/L ammonia water

The best sampling time for iodine analysis is after 14:00 PM

Cui et al., 2022 [61]

1) WMS, SLS, SMS and PS (unstimulated and stimulated)

2) Between 7:30 AM and 8:30 AM

- Healthy patients (control):

1) 40

2) 14M:26F

3) Mean age: 49.7 ± 3.7

- Diabetes mellitus (DM) patients:

1) 40

2) 14M:26F

3) Mean age: 50.1 ± 4.8

- Good oral hygiene on the day of sampling

- No eating, drinking, smoking or oral hygiene procedures 30 min before sampling

- Patients were asked to mouth-rinse with water right before sampling

- Salivary flow rate

- Salivary glucose

- Saliva was collected from all 80 participants using the following methods (5 mL total sample from all 6 methods):

1) Non-stimulated whole saliva (UWS); chewing non-coated Salivette® swab for 3 min

2) Stimulated whole saliva (SWS); chewing citric-acid-coated Salivette® swab for 3 min

3) Non-stimulated sublingual/submandibular saliva (USS); putting non-coated Salivette® swab under the tongue for 3 min

4) Stimulated sublingual/submandibular saliva (SSS); putting citric-acid-coated Salivette® swab under the tongue for 3 min

5) Non-stimulated parotid saliva (UPS); placing non-coated Salivette® swab near the left parotid duct for 3 min

6) Stimulated parotid saliva (SPS); placing citric-acid-coated Salivette® swab near the left parotid duct for 3 min

- At the end of all 6 methods, swabs were collected in pre-chilled polypropylene tubes placed on ice

All samples were centrifuged at − 20 °C and stored at − 20 °C

- DM patients had a significantly lower saliva flow rate than control

- Stimulated samples had a significantly higher saliva flow rate than non-stimulated samples

- Saliva glucose level:

1) DM patients: saliva glucose levels were significantly higher in non-stimulated samples; UPS > USS > UWS > > SPS > SSS > SWS

2) Control; NSD between different methods; USS > SSS > SPS > UPS > UWS > SWS

3) Saliva glucose levels were significantly higher in DM group compared to control in all 6 methods

- In conclusion for DM patients, stimulated methods had higher saliva flow rates while non-stimulated methods had significantly higher glucose levels

- The UPS (before breakfast) method, had the most correlated glucose level with blood glucose level and can serve as a non-invasive blood glucose monitoring for DM patients

Cornejo et al., 2022 [71]

1) WMS (unstimulated and stimulated)

2) NM

1) 11 (Healthy)

2) 4M:7F

3) Age range: 6 – 28 months

NM

- Salivary cariogenic streptococci count

- 2 different methods were assessed. Only 1 method was used for each patient:

1) Absorbent (unstimulated); a cotton swab was swabbed on the inner-cheek mucosa and floor of the mouth in figure of 8 motions until the cotton was completely soaked. Swabs were plated TYSCB containing Petri dishes, then placed in Eppendorf-type tubes containing PBS

2) Non-absorbent (stimulated) (1 mL); simulation was done by glove-covered fingers. Then stimulated saliva was collected from the floor of the mouth by aspiration with a plastic syringe into an Eppendorf-type tube

- Cultures were incubated at 36 ± 1 °C for 48 h

- Colony forming units (CFU/mL) were analyzed

- Mean rank of CFU/mL count:

Absorbent (unstimulated) (1.83) > > Non-absorbent (stimulated) (1.17)

- Mean rank of counting on cultures:

NSD; Absorbent (unstimulated) (1.54) ≅ Non-absorbent (stimulated) (1.46)

- S. sobrinus positive results (qPCR):

NSD; Non-absorbent (stimulated) (75%) > Absorbent (unstimulated) (36.4%)

- S. mutans positive results (qPCR):

Absorbent (unstimulated) (45.5%) > Non-absorbent (stimulated) (41.7%)

- The absorbent swab method was more effective in recovering microorganisms