1. Forensic Biology Screening Workshop
Saliva

2. Saliva Colorless fluid secreted by 3 glands in the mouth
Sublingual, submandibular, and parotid
Saliva from parotid glands contain amylases, enzymes, which aid in the digestion of carbohydrates
Saliva is composed of electrolytes, enzymes, mucus

3. Saliva
Screening for saliva is based on detection of high levels of amylase in the sample
It is not a confirmatory test; amylase is found in other body fluids
Serum, urine, sweat, lip mucous, semen, feces, etc.
The concentration of amylase in saliva is variable among individual; if amylase is not detected in a sample it does not mean saliva is not present

4. Saliva UV light can be used to aid in locating saliva stains
The intensity of the fluorescence can be affected by the substrate, concentration of the stain, and other body fluids
Saliva does not fluoresce as intensely as semen

5. Saliva Humans have both pancreatic and salivary amylase
Salivary amylase is a hydrolytic enzyme
Salivary amylase is also referred to as ptyalin

6. Saliva -amylase Found in humans elephants, rats, and pigs
Cleaves starch at its internal bonds (acts on  1,4 glucosidic linkages) allowing for compound hydrolysis-- the total breakdown of starch to maltose or glucose and dextrin
β-amylase
Found in plants
Cleaves only  1,4 glycosidic links
Can’t cleave starch at its internal bonds so there is an incomplete starch breakdown (maltose)

7. Amylase
One of the earliest tests for amylase was the starch-iodine test
Iodine solutions cause starch to turn a deep blue color
Amylase is a starch hydrolyzing enzyme
The presence of amylase causes the disappearance of the blue color (due to hydrolysis of the starch) and can be used an indicator for the presence of amylase

8. Saliva Both α and β-amylase react with starch iodine tests
β-amylase results in a hazy/cloudy clearing due to partial breakdown of starch
α –amylase has full clearing due to complete breakdown

9. Amylase Testing Limitations: Not confirmatory
Not specific for human amylase
Is specific for α-amylase
α-amylase can be found in other species

10. Starch Iodine Radial Diffusion Test
HOW TO MAKE:
Phosphate buffer, pH 6.9 :
NaH2PO4 , anhydrous 2.7g
Na2HPO4 , anhydrous 3.9
NaCl 0.2g
Dissolve in 500 ml of distilled water
Iodine development solution
KI 1.65 g
I g
Distilled water 30 ml
Dissolve by stirring for 5 minutes at 65°C in a fume hood. Store saturated I2 solution in dark stoppered bottle
Working solution in 1/50 dilution with distilled water

11. Starch Iodine Radial Diffusion Test
HOW TO MAKE:
Gel test plates (2% agarose, 0.1% soluble starch)
Phosphate buffer, pH ml
Agarose 0.2 g
Soluble starch 0.01 g
Heat to boiling and continue stirring constantly until all the agarose is dissolved. Divide gel solution and pour into 3-2" disposable plastic Petri dishes. Allow to polymerize completely.
Store gels inverted (to retard dehydration) at 4°C.

12. Starch Iodine Radial Diffusion Test
How to perform:
Extract a small piece of stained material with 50 µl distilled water
Note: run at least one positive control consisting of a known dilution of fresh liquid saliva (1/500 in distilled water) and a negative control consisting of distilled water
**some laboratories add a second positive control consisting of a 1/100 dilution of fresh saliva
Punch holes in gel plate with a vacuum pipette, leaving 1.5 cm between the sample wells
Place samples to be tested in the sample wells using a pipette. Each well holds approximately 4 µl of liquid

13. Starch Iodine Radial Diffusion Test
How to perform:
Cover the Petri dish and place in an incubator at 37°C for 6 hours or overnight
Stain the plate by pouring a 1:50 dilution of saturated iodine solution onto the surface. Rinse with distilled water
Clear circles around the wells indicate areas of amylase activity. The diameter of the clear circle is proportional to the square root of the concentration of amylase. Record the diameter and results in notes
Good idea to invert the plate in the incubator because this prevents the condensation that builds up in the plate from falling onto the testing surface.

14. Starch Iodine Radial Diffusion Test
How to perform:
A positive test is one in which the ring size is equal or greater in size than the positive control
**some laboratories assess the level if more than one positive control is run
An inconclusive result is one in which the ring size is less than the positive control but greater than the negative control
A negative result is an absence of any clear ring
There are interpretive issues with this test because a positive result may be overstating the value of the test. There are other body fluids that may also give a positive result. Each person has a differing level of amylase activity so even not having a clearing that is “positive” does not mean that saliva is not there. For example, there are many people at our lab who can not use their own saliva for their control because they will not get a clearing. When I make a 1:500 dilution my clearing is bigger than others, so I may call something inconclusive while someone else when compared to their control would be positive. You have to be very careful with interpretation of this test.

15. Amylase Diffusion Video

16. Phadebas ® Test Commercially available Blue starch polymer
Blue dye covalently attached to the starch and upon hydrolysis a product is obtained which is colorimetrically evaluated

17. Phadebas ® Test HOW TO MAKE:
Phadebas® tablets (commercially available)
0.5 M Sodium hydroxide (commercially available)
HOW TO STORE:
Follow instructions on product insert
Pharmacia/Upjohn # or Pharmacia Diagnostics, Piscataway, New Jersey)

18. Phadebas ® Test HOW TO PERFORM:
Place a small piece of the sample material in a 10 x 75 test tube. In a second tube, place an equal-sized piece of known saliva stain as a positive control. In a third tube add no sample (negative control)
Add 1.0 ml distilled water and ¼ Phadebas tablet to each tube using clean forceps
Vortex to mix thoroughly

19. Phadebas ® Test HOW TO PERFORM: Incubate at 37°C for 30 minutes
Add 0.25 ml of 0.5 M sodium hydroxide to each tube to stop the reaction
Centrifuge for 5 minutes

20. Phadebas ® Test Interpretation:
A transparent dark blue supernatant of equal or greater intensity than the positive control is regarded as a positive test for amylase activity
A blue color that is less intense than the positive control but darker than the negative control is considered inconclusive for presence of amylase
No blue color is considered negative for presence of amylase

21. Phadebas ® Test Positive test Negative test

22. Phadebas Video

23. Amylase Mapping HOW TO PREPARE:
Use one Phadebas tablet per 5ml of distilled water
Ten tablets/50ml distilled water for piece of 46x57cm Whatman 1 filter paper
Crush tablets using mortar and pestle
Mix the Phadebas tablets with distilled water and put in a sprayer
Note: Since the Phadebas mixture is actually a suspension, it must be kept mixed during spraying to evenly distribute the Phadebas particles

24. Amylase Mapping HOW TO PREPARE:
Hang or lay flat in a fume hood pieces of filter paper to be sprayed. Use gloves and mask to prepare papers
Spray the mist evenly onto the paper. Avoid spraying too heavy so that it does not run down the paper. Approximately 10ml per 900 cm2 gives a suitable covering
The paper can be used immediately or can be used dry
Note: If papers are made up ahead of time, stored in a dark dry place

25. Amylase Mapping HOW TO PERFORM:
If the paper is used right away, there is no need to rewet the paper. If paper is dry, re-spray the paper with distilled water until it is damp
Caution: When rewetting, if paper is sprayed too much or too hard the blue particles will puddle
The paper is then laid spotty side down on the item under examination and its position marked
A piece of plastic sheeting is laid over the filter paper and pressed for minutes (30 minutes at 37C)
Use of a flat board or sheet of glass on top of sheeting can aid in this process
Crush Phadebas tablets with a mortar and pestle – the mixture is a suspension and you need to keep the solution mixed to evenly distribute the Phadebas particles.
You can make these up in advance and let dry – you just need to rewet the paper prior to use with deionized water.
You can use a sheet of glass or a wood board to apply pressure – needs to have good, even contact.
This method is useful on larger items or items where fluorescence is not able to be observed due to the substrate

26. Amylase Mapping HOW TO PERFORM:
After incubation, the paper is removed and dried. Positive areas appear as pale blue zones in place of the mottled blue negative areas. Slight shrinkage of the filter paper may occur during drying
NOTE: The paper (while still damp) can be sprayed with acid phosphatase mapping reagents to search for semen stains.

27. SALIgAE® Test Test available from Abacus Diagnostics
Sensitive, simple, and reported to be more accurate than other tests
Solution in a tube
Changes color with addition of an extract containing saliva
Exact mechanism – proprietary
Sensitivity - ~1:1000
Sensitivity results per validation paper – source at end of presentation. Not per SALIGaE insert – was not noted on insert
Still not a confirmatory test

28. SALIgAE® Test HOW TO MAKE: SALIgAE ® Test kit (commercially available)
HOW TO STORE:
Follow instructions on product insert

29. SALIgAE® Test HOW TO PERFORM:
Place approximately 5 mm2 cutting or ½ of a swab into a sterile 1.5 ml microcentrifuge tube
Place an equal-sized piece of known saliva stain as a positive control in a separate sterile 1.5 ml microcentrifuge tube
In a third tube add no sample (negative control)
Pipette 30 µl – 50 µl of sterile deionized water into the tube

30. SALIgAE® Test HOW TO PERFORM:
Incubate for 30 minutes at room temperature
Allow the test vials to warm to room temperature
Remove bubbles from the test vials by gently tapping the vials
Add 8 µl of sample to the test vial
Mix gently

31. SALIgAE® Test HOW TO PERFORM: Read the result after 10 minutes
A yellow color change indicates a positive result
No color change indicates a negative result
A negative result indicates that there is no saliva present or is below the limit of detection of the test.

32. SALIgAE® Test

33. SALIGaE Video

34. Rapid Stain Identification (RSID®) of Saliva
Reaction:
“S” area – antihuman salivary amylase mobile monoclonal antibody conjugated with colloidal gold
If human salivary amylase is present in the sample, a mobile Antibody-Antigen complex is formed and travels to the “T” area
“T” area - antihuman salivary amylase stationary monoclonal antibody

35. Rapid Stain Identification (RSID®) of Saliva
Reaction:
Stationary antibodies in the “T” area catch the mobile Antigen-Antibody complex
Forms Antibody-Antigen-Antibody complex
When the antibodies aggregate, a red line is formed
“C” area is internal control
Binds mouse antibodies with anti-mouse IgG which ensures the fluid transported the length of the strip and the test is working properly

36. Rapid Stain Identification (RSID®) of Saliva
HOW TO PERFORM:
Place a small cutting of the stain into a 1.5 ml microcentrifuge tube
Add µl RSID Extraction Buffer
Incubate for 1-2 hours at room temperature
Remove 20 µl of extracted sample and add it to 80 µl of RSID TBS Running Buffer in a new tube
Add the total 100 µl to the sample well of the RSID® card
Read result after 10 minutes

37. Rapid Stain Identification (RSID®) of Saliva
HOW TO PERFORM:
Positive result
Test line and control line are both present
Negative result
Only control line is present
Invalid result
No line at all or only test line is present (control line absent)

38. Rapid Stain Identification (RSID®) of Saliva
LIMITATIONS:
False positives with breast milk, fecal material, and vaginal fluid
False negative – high dose hook effect
Sample containing up to 50 µl saliva did not result in high dose hook effect
If high dose hook effect is possible, dilute using 1:100 dilution of sample