MY IAOMR ESSAY

1. INTRODUCTION:
Saliva was once a largely unheralded and ignored secretion.But in the recent years
the diagnostic use of saliva has attracted the attention of numerous investigators.
Salivary diagnostics is a dynamic and emerging field utilizing nanotechnology and
molecular diagnostics to aid in the diagnosis of oral and systemic diseases. Saliva is a clinically
informative, biological fluid that is useful for novel approaches to prognosis, laboratory or
clinical diagnosis, monitoring of patients with both oral and systemic diseases.As the pursuit of
timely, cost effective, accurate and noninvasive diagnostic methodologies is an endeavour of
urgency among clinicians.It has laid down foundations for the field of salivary diagnostics.
TYPES OF SALIVA:
a)Gland specific:
The collection and evaluation of the secretions from the individual salivary glands.It
is primarily useful for the detection of gland specific pathology (ie) infection and obstruction .
b)Whole saliva:
mixture of oral fluid which includes :
I. Secretions from both major and minor salivary glands
II. Several constituents of non salivary glands such as GCF
III. Expectorated bronchial and nasal secretions
IV. Serum and blood derivatives from oral wounds
V. Bacteria & their products,viruses and fungi
VI. Desquamated epithelial cells ,cellular components ,food debris

2. Counterbalance in salivary diagnostics
PRECAUTIONS TO BE TAKEN BEFORE SAMPLE
COLLECTION:
Avoid
 Alcohol for 12 hrs. & dairy products for 20 mins. before sample collection.
Advantages:
1.Can be collectednon
invasively.
2.Simple equipment
and cost effective.
3.Minimallytrained
individualsrequired.
4.It is easyto handle
than bloodasit doesnot
clot .
5.Minimal riskof
contractinginfections.
6.Easy to store and
transport.
Disadvantages:
1.Prone for diurnal
variations.
2.Notalwaysreliably
reflectthe concentrationsof
these moleculesinserum.
3.Sensitivedetection
systemsare needed.
4.containsanalytesin
concentrationsthatare
1000-fold lessthanthose in
blood.
Figure1

3.  Foods with high sugar or acidity, or high caffeine content before sample collection.(may
lower ph or increase bacterial growth )
 Do not eat a major meal within 60 minutes before sample collection.
 Document consumption of alcohol,caffeine,nicotine& any medication usage.
 Wait at least 10 minutes after rinsing before collecting saliva to avoid sample dilution.
BLOOD CONTAMINATION OF SALIVA:
 Even an invisible amount of blood contamination has the potential to falsely elevate
salivary analyte levels.
 Should not brush teeth within 45 minutes .
 Dental treatment should not be performed within 48 hours prior to sample collection.
METHODS OF COLLECTING WHOLE SALIVA:
I. Draining
II. Suction
III. Spitting
IV. Absorbent (sponge ) method
Type of saliva Type of collection Methods Technique
Whole saliva unstimulated Passive drainage Saliva flows
passively from the
mouth into a
graduated container
Whole saliva unstimulated Active drainage The saliva
accumulated in the
mouth must be
regularly
expectorated into a
graduated container
Whole saliva unstimulated suction Saliva pooled on the
mouth floor is
vacuumed and
accumulated in a
graduated container
Whole saliva unstimulated absorption Swab,cotton,or
gauge roll,Pre-
TABLE1

4. LASHLEY CUP / CARLSON – CRITTENDEN COLLECTOR:
(FOR PAROTID GLAND)
It is used for parotid. It has 2 metal cups with 2 outlet tubes. Inner cup collects
saliva. Outer cup is connected to vacuum. Basic design was modified by Lashley which has a
single cup with concentric chambers.
The inner chamber can be positioned over parotid duct orifice for collection of
saliva.While suction is applied to outer chamber sample collection takes place. Parotid saliva can
be stimulated by applying 1ml of 2% citric acid to dorsum of tongue every 30 sec. Depending on
age & health status of subject flow rate is usually 1 to 3 ml / min.
SALIVARY DIAGNOSTICS IN ORAL DISEASES:
Saliva in dental caries:
Saliva may also be used for the monitoring of oral diseases. Anaerobic bacteria can
survive in saliva, and can utilize it as a growth medium. Studies have shown that salivary
parameters such as flow rate, viscosity, ph, buffering capacity were decreased in subjects with
weighed,is inserted
into the mouth for a
certain amount of
time and later
weighed again
Whole saliva stimulated Mechanical Paraffin,silicone,or
unflavoured
chewing gum
Whole saliva stimulated Gustatory Applying 2%citric
acid to the lateral
borders of the
tongue
Glandular Method
Glandular saliva Parotid 1.Micropipette,Lasley’s vacuum
chamber,Carlson-Crittenden cups
Glandular saliva Submandibular
sublingual
1.Cannulation of the duct with
micropippete
2.Another technique is to block off
secretion from parotid gland with absorbent
swabs
Glandular saliva Labial&palatal
glands
1.Filter paper disc
2.Disc of other synthetic materials

5. high risk of caries. So saliva testing is an important part in routine protocol while diagnosing and
treating dental caries.
saliva in periodontal disease:
Saliva is a fluid that contains locally derived and systemically derived markers of
periodontal disease.
Salivary
biomarkers
specific
secretory
IgA2,IgG,lysozyme,
peroxidase,Ca++,β
-glucuronidase, β-
glucosidase,butyra
te esterase
,cystiene
aminopeptidase non specific
proteins,MG1and
MG2),lactoferrin,
histatin,
fibronectin,
proline,Creactive
proteinINFLAMMATORY
PGE2,IL-1β,
IL-6,TNF-α,
MMP-8,MMP-9,
MMP-13,
Telopeptide,
osteocalcin,osteop
ontin
Emerging
markers
salivaryproteome
salivary
transcriptome
Category Salivary biomarkers Strength
Salivary biomarkers in
periodontal disease
TABLE2
FIGURE2

6. (correlation with clinical parameters;S -strong;P-potential;Q –questionable)
Saliva from treated periodontitis patients had higher IgA and IgG levels to
periodontal pathogens porphyromonas. Gingivalis and Treponema. Denticola than as compared
to saliva from control subjects.
Circulatory C-reactive protein may reach saliva via GCF or the salivary glands.
High levels of C-reactive protein have been associated with chronic and aggressive periodontal
diseases.
Category Salivary biomarkers Strength
Proteins IL-1β S
MIP-1α S
Arginase S
Soluble CD14 P
IFN-γ and
IFN-γ/IL-22 ratio
P
Lactoferrin P
Dipeptidyl peptidase P
Chemerin P
Procalcitonin P
Calprotectin P
Myeloperoxidase P
IL-1β P
TLR4 P
Β-glucuronidase P
CRP P
IL-6 Q
IL-8 Q
TNFα Q
Metabolites Nitric oxide S
β-
hydroxydeoxyguanosine
S
Platelet activating factor P
Neopterin P

7. Sjogren’s syndrome:
Sialochemistry of the collected glandular saliva samples may show several
characteristic changes in electrolytes and enzymes in sjogren’s syndrome ,reflecting the effect of
autoimmune attack on the secretory cells in individual salivary glands.
A consistent finding is increased concentrations of sodium and chloride. This
increase is evident in both whole and gland-specific saliva.
In addition, elevated levels of IgA, IgG, lactoferrin,albumin a decreased
concentration of phosphate were reported in saliva of patients with SS.
Increased β2 microglobulin, lipid levels, cystatin C and S levels,inflammatory
mediators (ie.)eicosanoids,PGE2, thromboxane B2, interleukin-6.SS anti-La antibodies were
primarily found in the saliva of patients.
Since no single salivary or serum constituent can accurately serve as a diagnostic
marker for SS,the most important aspect of the salivary diagnosis for this disease is evaluation of
the reduced quantity of saliva.
saliva in oral candidiasis :
Saliva can be used for the detection of oral candidiasis and salivary fungal counts
may reflect mucosal colonization.
Recurrent aphthous stomatitis :
Initial phase of SS
autoimmune attack on
secretory cells precede
salivary gland dysfunction
sialochemistry is often
useful
Severe salivary gland
dysfunction
autoimmune inflammation
has resulted in massive loss
of active secretory cells
sialometry is highly
diagnostic
FIGURE3

8. Evidence shows that there is increased oxidative stress.Levels of both plasma
and salivary antioxidants are decreased in patients with RAU.
SALIVA IN SYSTEMIC DISEASES:
Some systemic diseases affect salivary glands directly or indirectly, and may
influence the quantity of saliva that is produced, as well as the composition of the fluid.The
characteristic changes may contribute to the early detection of these diseases.
CVS disorders:
If salivary amylase levels are low (<52U/L)pre operative undergoing heart
surgery there is increased risk of mortality.
PLASMA Vs SALIVA BIOMARKERS
Salivary C-Reactive protein levels in healthy individual is
But CRP remains a non-specific inflammatory response factor in many conditions
including periodontal disease.
Copeptin,
MR-proADM,
homocysteine,
h-FABP,sLOX-1,
sST2,ANP,GGT,
Lp-PLA2,CgA,
procalcitonin,
choline,NPs,
myoglobin
adiponectin,
irisin,SAA,
ischemia-modified albumin,
monocyte
chemoattractant
protein 1,
sICAM-1,
GRO-α
Plasma Saliva
CRP,
CK,
CD40
ligand,
MPO,cT
n(I and
T),MMP
0.12-10ng/ml
FIGURE4

9. SalivaryMarkers combined with an ECG correlates well with myocardial infarct
patients.
Robust clinical studies are required to validate salivary biomarkers for CVD
CHRONIC RENAL FAILURE:
Colorimetric strips were used to monitor salivary nitrate and uric acid before and
after hemodialysis.It has been suggested that a salivary test could be used by patients to decide
when dialysis is required,thereby eliminating unnecessary visits to a dialysis clinic.
Category Components
Electorolyte salivary urea(Normal=31.9mmol/l)
(>128mmol/l)
salivary creatinine (Normal = 6-18mmol/l)
(>85mmol/l)
Metabolite Salivary phosphate
cortisol
nitrite
uric acid
sodium
chloride
pH
amylase and
lactoferrin.
Salivary nitrate is excellent biomarker when compared to serum nitrate.
Diabetes:
Because of the large diabetic population,combined with the current epidemic of
type-2 diabetes,an oral test to monitor blood glucose would be highly desirable.
Unfortunately,while it is relatively easy to measure salivary glucose,due to the
multiple sources of this material in the oral cavity,salivary glucose levels do not correlate
with blood glucose levels.

10. Insulin can be detected in saliva.A positive correlation between saliva and serum
insulin levels following a GTT was reported for healthy subjects, NIIDM patients.
Salivary insulin levels reached maximum 30 minutes after serum levels.
Sarcoidosis:
As a part of systemic sarcoidosis, granulomatous foci may be seen in the
salivary glands,which may even cause bilateral swelling of the parotid gland.The flow is
elevated.Sialochemistry fails to reveal inflammatory changes.
Kallikrein is reported to be low.While angiotensin converting enzyme (ACE)
will be elevated in the saliva. Normally,this enzyme is absent in saliva.
Cystic fibrosis:
Cystic fibrosis is a genetically transmitted disease of children and young adults
which is considered a generalized exocrinopathy.
Source of salivary sample Sialometric report Sialochemistry
(Elevated levels of)
Submandibular gland
Minor salivary glands
Decrease in flow rate
Electrolytes:
sodium,
calcium,
(causes turbidity)
Metabolites:
proteins,
neutral lipids,
phospholipids,
glycolipids
Biomarkers:
Abnormal
epidermal growth factor,
PGE2
Parotid gland No characteristic
changes
No characteristic
changes
TABLE3

11. MALIGNANCY:
Salivary analysis may aid in the early detection of certain malignant tumours.
p53 is a tumour suppressor protein which is produced in cells exposed to
various types of DNA-damaging stress.Inactivation of this suppressor through mutations and
gene deletion is considered a frequent occurrence in the development of human cancer.
Defensins are peptides which possess antimicrobial and cytotoxic
properties.They are found in the azurophil granules of PMNs.
conditions Comparison
carried out
Markers/analytes Correlations
observed
Remarks
Head and neck
cancer
Saliva-tumour
tissue
Mitochondrial
DNA
Positive Increased levels
that decreased
in post-surgical
condition
Oral SCC Saliva-serum P53 antibody
Anti-defensin1
Positive Saliva may offer
a specific
method for
detection of a
subset of oral
SCC with p53
aberrations
Malignant
ovarian tumours
Saliva-serum CA 125 Positive Saliva assay
showed better
assay value than
serum
Breast cancer Saliva-serum Cellular
erythroblastosis
oncogene-B-2
(c-erbB-2)
Positive ErbB-2 protein
may have
potential use in
the initial
detection and/or
follow-up
screening of
breast cancer in
women
TABLE4

12. HORMONE LEVELS:
Salivary hormone levels represent the non-protein bound (free) serum hormone
levels.
Salivary Hormone Diagnostic value Correlation with serum
concentrations
(using correlation ratio)
Cortisol 1.Identifying patients with
cushing’s syndrome and
addison’s syndrome
2.monitoring hormone
response to physical exercise
and acceleration stress
r=0.36
Aldosterone Patients with primary
aldosteronism(Conn’s
syndrome)
r=0.60
Testosterone and
dehydroepiandrosterone
Aggressive behavior and
athletic activities
Progesterone Prediction of ovulation r=0.47-0.58
Estradiol Assessment of feto-placental
function(decreased levels
suggest fetal growth
retardation)
r=0.56
Insulin Identifying diabetic patients r=0.58 (appears within 30
minutes in saliva)
Increased salivary
estriol:progesterone ratio
Predictor of pre-term labour _
TABLE5

13. INFECTIOUS DISEASES:
Pathogen/disease Salivary biomarkers Salivary
antibodies
Sensitivity and
specificity
Helicobacter pylori MUC-5B,MUC7, Bacteria itself (by
PCR assay )
IgG antibody 84% (more
sensitive than
feces)
Shigellosis Anti lipopolysaccharide Anti-shiga
toxin antibody
78%
Lyme disease
(Borrelia
burgdorferi)
_ Anti-tick
antibody
Sensitivity 96%
Specificity98%
Hepatitis B HbsAg,HbsAb,HbcAb,DNA IgM
antibody(by
orasure-oral
mucosal
transudate
Sensitivity98.7%
Specificity99.6%
(Correlated with
serum antibody
levels)
Hepatitis C RNA IgG Sensitivity 98.7%
Specificity99.6%
Human Herpes
virus
8,cytomegalovirus,
Epstein barr virus
Shedding of viral DNA(by PCR) _
81%sensitivity
81%specificity
HIV P24 antigens ( by
ORAQUICK)
IgA anti HIV
antibodies(by
ELISA and
western blot)
100%sensitivity
100%specificity
HSV1 HSV-1 virus by PCR assay _ 94%sensitivity
94%specificity
measles _ IgM antibodies 97% sensitivity
100%specificity
Mumps Elevated salivary
Amylase(>52U/L)
IgM antibodies 94%sensitivity
94%specificity
rubella _ IgM antibodies 98%sensitivity
98%specificity
TABLE6

14. SALIVA IN PSYCHIATRY:
Saliva has used to detect pathological anxiety by measuring salivary
levels of 3-methoxy-4hydroxyphenyl glycol(MHPG) and substance-P
elevated
Ebola virus IgG,RNA,Antigen 78%sensitivity
Oral candidiasis Hsp70,calprotectin,histatins,mucins,
peroxidases
100%sensitivity
100%specificity
dengue _ Anti-dengue
IgM and
IgG(IgG
differentiates
primary from
secondary)
92%sensitivity
100%specificity
Hypothalamus
Pituatory gland
DHEACortisol
stress
Ratio
ACTH
Adrenal glands
Catabolic function
Stress hormone
Anabolic function
Anti-stress hormone
MHPG,
Substance-p
Released into saliva
FIGURE5

15. DRUGS:
Passive diffusion across a concentration gradient accounts for the
appearance of a drug in saliva.Therefore,drugs which are not ionizable, or not ionized within
the ph range of saliva, are most suited to salivary drug monitoring.
Saliva based surveillance for specific drugs
Drug
category
Drug Technique used Reported
levels
(if
available)
Correlation
with serum
concentrations
(using
correlation
ratio)
Antiepileptic Levetiracetam High performace liquid
chromotagraphy/electrospray
tandem mass spectrometry
5.6-27.6
µg/ml
r=0.83
Carbamazepine Fluorescence polarization
immunoassay
r=0.89
Phenobarbital
and phenytoin
Fluorescence polarization
immunoassay
r=0.89
Immuno-
suppressant
cyclosporine Radioimmunoassay 8.3 ± 5.2
µg/ml
r=0.84
Respiratory Theophylline Apoenzyme reactivation
system and fluorescence
polarization immunoassay
r=0.85
Anti-cancer Cisplatin
Carboplatin
Platinum levels by atomic
absorption spectrometry
0.13
mg/ml
1.15
mg/ml
r=0.89
r=0.89
Diagnostic utility of saliva in evaluating illicit drug use
category Specific analytes Principle methods Minimal
detectable level
Metabolites of Cotinine(major nicotine ELISA/EIA 0.15 ng/ml
TABLE7
TABLE8

16. substance abuse metabolite)
Thiocyanate(cigarette
smoking)
Fourier trans-form
infrared spectroscopy
0.83 mmol/ml
D-9-
Tetrahydrocannabinol(a
major psychoactive
component of
marijuana)
Qualitative YES or NO
assay
( for atleast 4 hours
after marijuana is
smoked )
25 ng/ml
Recreational drugs cocaine Qualitative YES or NO
assay
20 ng/ml
Pharmaceutical
drugs used as
recreational drugs
Amphetamines Qualitative YES or NO
assay
25 ng/ml
Barbiturates Gas chromatography 0.1-1µg/ml
Benzodiazepines
Lorazepam
Clonazepam
Alprazolam
midazolam
High performace liquid
chromotagraphy/tandem
mass spectrometry(LC-
MS/MS)
1.5 ng/ml
1.5 ng/ml
1.5 ng/ml
2.2 ng/ml
Opiods Qualitative YES or NO
assay
25 ng/ml
Phencyclidine High performance LC-
MS/MS
1 ng/ml
BONE TURN-OVER BIOMARKERS:
Bone turn-over markers Positive relation: Deoxypyridinium(D-PYR)
Osteocalcin
Hepatocyte growth factor
IL-1β
Negative relation:Osteonectin
Alkaline phosphatase(ALP)
SALIVA IN FORENSICS:

17. Saliva test have been used for a wide variety of forensic
studies.Samples can be obtained from drinking glasses,cigarette butts,envelopes,and other
sources and then used to detect blood-group substances or salivary genetic proteins(primarily
proline rich polymorphisms).Saliva is often present at crime scenes,along with other body
fluids,and since DNA is relatively stable in the dry state,the samples can be used to detect the
criminal.
SALIVARY PROTEOMICS:
Proteomics is analysis of the portion of the genome that is expressed.By
using two-dimensional gel electrophoresis/mass spectrometry and “shotgun” proteomics
approaches collectively,1166 salivary proteins have been identified: 914 from the parotid
fluid and 917 from the combined submandibular and sublingual fluids.
SALIVARY TRANSCRIPTOMICS:
Salivary transcriptome is an emerging concept.They included mRNA
molecules that cells use to convey the instructions carried by DNA for subsequent protein
production.This discovery opened a door to new avenue of salivary transcriptomic
diagnostics.
Li et al. found that RNA molecules elevated in oral cancer tissues are
also elevated in saliva,which prompted them to examine the scope and complexity of RNA
present in saliva.
Other research groups,particularly from forensic sciences,are focusing
on multiplex mRNA profiling for the identification of body fluids including saliva.
CONCLUSION:
Saliva offers an alternative to serum as a biologic fluid that can be
analysed for diagnostic purposes.Nevertheless,levels of certain markers in saliva are not
always a reliable reflection of the levels of these markers in serum.Changes in salivary flow-
rate may affect availabilityofsalivary markers.Despite the limitations,the use of saliva for
diagnostic purposes is increasing in popularity.However,before a salivary diagnostic test can
replace a more conventional one,the diagnostic value of a new salivary test has to be
compared with accepted diagnostic methods.Potential advantages of salivary analysis for the
diagnosis of systemic disease suggest that further studies are warranted.
REFERENCES:

18. 1)Diagnostic potential of saliva:current state and future applications by Tina Pfaffe,
(clinical chemistry57:5;687)(2011)
2) The diagnostic applications of saliva-a review by Eliaz Kaufman and Ira B.
Lamster.Crit Rev oral Biol Med.13(2):197-212(2002)
3) Point-of-care diagnosis of periodontitis using saliva:technically feasible but
still a challenge.Front.Cell.Infect.Microbiol.,03sept2015
4) Clinical and diagnostic utility of saliva as a non-
invasive diagnostic fluid:a systematic review.Biochemica Medica 2015;25(2):177-92
5) Diagnostic potential of saliva:current state and future
applications by Tina Pfaffe(clinical chemistry57:5;675-687) (2011)
6) Saliva:a diagnostic biomarker of periodontal diseases.journal of Indian
Society of Periodontology 2011;15(4)by Priti Basgauda Patil
TABLE1
TABLE4 TABLE7 TABLE8
TABLE5 TABLE6
TABLE2
FIGURE2
A.Selva arockiam,
CRI,
CSI College of Dental Sciences and
Research,
Madurai,
Tamilnadu