FORENSIC EXAMINATION OF ITEMS FOR THE
PRESENCE OF SALIVA
DESCRIBED TEST
Phadebas® Forensic Press test
Background
The enzyme α-amylase is found in very high levels in saliva. Its activity in stains is used as an
indicator for the presence of saliva. The test used to identify amylase uses Phadebas®, consisting
of starch microspheres with a blue dye cross-linked to the starch. The blue starch microspheres
are immobilised on filter paper sheets. In the presence of amylase the starch is digested, releasing
the water soluble dye, which diffuses through the pores of the filter paper. The resulting blue
colour is visually observed on the non-reagent side of the Phadebas® paper.
The Press test is performed when it is necessary to localise an amylase positive area on an item. If
a very strong reaction is obtained with the Press test and there is no other obvious contaminating
material, this is interpreted as an indication of saliva.
Although the Phadebas® test is specific for amylase, it is not specific for saliva. Amylase is found
in other body fluids, although normally at much lower levels than in saliva. Generally, amylase
found in other body fluids will not be present in sufficient quantity for detection by the Press test
method. As a reference on the differences in amylase activity between saliva and other fluids, the
below list was compiled from the results published in reference 1*:
Saliva: 263000 to 376000 IU/L
Urine: 263 to 940 IU/L
Blood: 110 IU/L
Semen: 35 IU/L
Nasal secretion: Undetectable levels
Sweat: Undetectable levels
When positive results are obtained on an item, it is recommended that a substrate control sample
also is submitted for DNA analysis at the same time, with the exception of swabs.
*
P.H. Whitehead, Ann E. Kipps; J. Forens. Sci. Soc. (1975), 15, 39-42
Phadebas® Forensic Press test protocol
1. As a positive control, and before testing the item, tear a corner from the Phadebas® paper and
envelop a fresh (damp) saliva-containing swab within it. Record the time that a positive
reaction is first observed. Note: If no colour develops within 5 minutes take another corner
and repeat using a different saliva donor. If again, no colour change is observed within 5
minutes, discard the Phadebas® paper and repeat using a fresh piece and a third saliva donor.
2. Place the item to be tested on a flat surface. The item can for example be stretched out over a
piece of glass board, covered in a plastic wrap.
3. Using a spray bottle, dampen the item with water.
4. Place a piece of Phadebas® paper over the area to be tested, with the blue reagent side in
contact with the item. On the non-reagent side, write the case and item number, date and
initial.
5. Spray water liberally onto the Phadebas® paper. The paper must not dry out during
the test period but it also should not be too wet.
6. Trace a rough outline of the area being tested on the Phadebas® paper so that it can be
accurately placed back on the item at a later time if necessary.
7. Lay another (plastic-wrapped), clean glass board on top of the paper and place a weight on
top of the glass. Use a 4 kg or heavier weight, to ensure good contact between the item and
the paper and always use the same weight for all Press tests.
8. At this point start a timer.
9. The test is to be observed for a time period of maximum 40 minutes. Record in the
examination notes the time that (a) positive reaction(s) is/are first observed. As well, record
the progression of the test frequently for the first ten minutes, and then at 5-minute intervals
until 30 minutes has elapsed, and finally at 40 minutes. Note: If a strong positive reaction has
occurred then the test may be stopped before 40 minutes, depending on the circumstances
(consult reporting scientist).
10. Record in the examination notes the time that any positive reaction(s) is/are first observed.
Also note the dimensions and intensities (e.g., weak, moderate or strong) of any amylase
positive areas observed during this time. Note: A positive is identified as a distinct area of
diffuse blue colour on the non-reagent side of the paper.
11. Remove the glass board leaving the Phadebas® paper in contact with the item.
12. Mark the boundaries and any garment markings, such as seams, directly on the Phadebas®
paper using an appropriate pen.
13. Using a wooden applicator stick, outline any positive areas by piercing small holes around the
area and, using a suitable pen, dabbing ink onto the fabric.
14. The Phadebas® paper can now be removed and hung to dry. Negative press papers can be
discarded but positive press papers must be retained with item. If a positive area is to be
submitted for DNA analysis, cut an approximately 1cm x 1cm portion of the material and the
corresponding piece of the Phadebas® paper into a labelled tube. An unstained area from the
item and the corresponding piece of the Phadebas® paper may also be submitted as controls.
15. In cases where the DNA analysis yields a low-level poor profile or if the profile is a complex
mixture where you cannot derive a major contributor, you may try to submit another piece
from the positive area of the Phadebas paper only for DNA analysis. This has been known to
sometimes yield an interpretable DNA profile.
Important: If the same area of the item must be checked for acid phosphatase activity, it can be
done once testing for amylase is complete. Apply the acid phosphatase reagent directly to the
Phadebas® paper. The paper can be replaced on the item and the potential semen stain delineated
(as above) and excised as necessary.
False Positives
In-house testing at several independent forensic laboratories has determined that no other
forensically relevant body fluid (sweat, semen and vaginal secretion) will react within 10 minutes
using the current protocol, even after repeated deposition. The exception is faecal stains that may
contain levels of amylase as high as those found in saliva. For this reason positive observations
within areas obviously contaminated with faeces should not be interpreted for the presence of
saliva. The presence of potential faecal material on an item should be recorded in the examination
notes.
False Negatives
Amylase activity within and among individuals can vary and it may be possible that a saliva
containing stain may not react, even after 40 minutes.
