Med. Sei. Law (1976) Vol. 16, No. 1
37
The Identification of Saliva in Stains in Forensic Casework S. J . BAXTER. BSC. PhD B. REES. BSC. PhD Home Counties Forensic Science Aldermaston, Reading
Laboratory,
INTRODUCTION The evidential value of saliva in stains encountered in crime investigation can be of considerable importance. Whether the stains are a result of expectoration or a consequence of some sexual or violent act resulting in saliva being mixed with other body fluids, the practising forensic scientist is faced with two fundamental problems: (1) localization of the stain, and (2) identification of the stain. In practice these two aspects arc often closely interlinked. Visualization of saliva stains is problematical. Unlike blood which has its own internal marker, i.e. haemo globin, saliva h u no luch readily visible constituent. In this respect it is similar to semen. The introduction of the acid phos phatase test for the location of semen stains has, however, overcome these problems. Kind (1964) has reviewed the acid phos phatase test for the identification of semen. As yet no test which compares with the acid phosphatase test for the localization of semen has been devised for saliva, although the use of Procion Red Μ χ 2B amylopectin for the visualization of amylase activity promises to parallel the acid phosphatase test in this respect (Whitehead, 1974). The literature on the identification of saliva stains is not extensive. The use of the amylase activity of saliva for its identification has been described by Nickolls (1956). Nelson and Kirk (1963) dealt more compre hensively with the possible markers for the identification of saliva. Human saliva is a very complex fluid but there appear to be 4 components which can be considered to offer
possible parameters for its identification. These are: 1. Enzymes (α-amylase, alkaline phos phatase) or proteins with specific activity such as lysozyme. 2. Non-cnzymic proteins—mucoproteins. 3. Buccal epithelial cells. 4. Inorganic components (nitrite and thiocyanate). Although there would appear to be few restrictions on the number of possible markers .for the identification of saliva, in practice there are considerable problems. As a consequence the identification of saliva stains and saliva in mixed stains has mainly relied on the amylase activity of saliva as detected by starch hydrolysis allied with microscopy for buccal epithelial cells. Other constituents which offer possible means of characterizing saliva are such that for practical purposes they have not found wide usage. Thus, the value of alkaline phosphatase, nitrite and thiocyanate in establishing the salivary origin of suspect material has been fully described by Nelson and Kirk (1963) and the authors are in agreement with their evaluation. Similar criticisms can be used against the use of lysozyme as a possible means for the identi fication of saliva. Thus, although this protein can be readily identified by immunological procedures, its wide occurrence in other body fluids such as semen and lacrimal fluid renders this of little value. T h e value of amylase as a means for detecting saliva is still of prime importance and will be discussed later.
38
Med. Sei. Law (1975) Vol. 15, No. 1
This paper reports work carried out in this laboratory which is concerned with bio chemical methods for the identification of saliva in stain and mixed body fluid stains.
buffer in the upper compartment of the extraction device illustrated in Fig. 1 . After this period the extraction device is centrifuged and the stain extract can then be removed from the lower compartment. This
MATERIALS Amylose Azure was obtained from Calbiochem Limited, 10 Wyndham Place, London. Barbitone buffered saline (sterile) p H 7-2 5x concentrated was obtained from Wellcome Reagents, Beckenham, Kent. Soluble Starch Analar grade was obtained from B D H Chemicals Limited, Poole, Dorset. METHODS The only satisfactory procedure for the identification of saliva in stains, of which the authors are aware, relies upon the high amylase activity of saliva. Thus, there are two methods whereby starch hydrolysis can be detected. The first of these relies upon the detection of unhydrolyzed starch, the second depends on the detection of products formed as a result of substrate hydrolysis. These two methods are dealt with separately. 1 . Detection o f Unhydrolyzed Starch Nickolls (1956) and Nelson and Kirk (1963) described methods which utilize the detection of unhydrolyzed starch for the identification of salivary amylase. The routine procedure used in this laboratory for the identification of salivary amylase in stains is a refinement of these techniques. This technique involves serial dilution of the material under investigation prior to incubation with soluble starch. In this method doubling dilutions up to 1 : 256 are prepared from salivary stain extracts. The details of the procedure are as follows. After localization of the suspected areas of saliva staining as much of the stained area as can be afforded, without prejudicing other test procedures, is removed from the garment. In the case of neat, uncontaminated saliva stairts ^ sq in of stained material is sufficient. Proportionately more is required in cases with smeared saliva stains or mixtures with other body fluids. The stained area after removal is extracted for J hr in 7 drops of
Fig. J: Extraction device. A, upper polystyrene tube 36 mm χ 6 mm; B, transparent adhesive tape snap-joint; C, small hole; D, lower polystyrene tube 36 mm χ β mm.
normally results in approximately 6 drops of stain extract being recovered. These are then treated as follows. Three drops of the extract are removed and heated in a sealed glass tube at 100° C for 10 min. T w o drops of the initial extract plus 2 drops of the boiled extract are then separately and serially diluted in glass tubes to dilutions of 1 : 256 using the same buffer as used for the stain extraction. T o each of these dilutions is added 1 drop of 1 per cent soluble starch. All tubes are then incubated at 37° C for 1 hr after which time 1 drop of iodine solution is added. Any
Baxter and Rees: Identification of Saliva
unhydrolyzed starch will be detected by the characteristic intense blue starch/iodine reaction. It is essential to treat a control area of cloth in precisely similar fashion.
39
the techniques described. Therefore, the presence of saliva in mixed stains with these body fluids cannot be satisfactorily established using this procedure.
Table I: Reactions of Diluted Saliva Stain Extracts with 1 per cent Soluble Starch using Starch/Iodine Colour Reaction as Indicator of Degree of Starch Hydrolysis
Extract Dilutions
Colour Reaction with Iodine after 1 hr Incubation at 37°C with 1 drop 1 per cent Starch Solution Neat 1 : 2 1 :4 1:8 1 :16 1 :32 1 :64 1 :128 1 :256
Extracted saliva stain
Untreated extract Boiled extract
—
Extracted control area
Untreated extract Boiled extract
— —
+ + +
+
+
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ = deep blue colour; + = faint blue colour; — = no blue colour.
Using this method an indication of the amylase activity in the stain extract together with its serial dilutions is obtained. In normal saliva stains complete starch hydrolysis occurs in the diluted extract at dilutions of 1 : 64 and sometimes to 1 : 128. In the boiled extract starch hydrolysis should not occur at all as the enzyme will have been heat inactivated. It does occur, however, that non-speciñc contaminating materials in the cloth can prevent the starch/iodine colour reaction so giving rise to false positive reactions unless adequate control procedures are taken. These false positive reactions rarely extend as far as the 1 : 8 dilution, however, indicating that this is caused by some agent in the cloth whose action is not enzymic. Typical results for such a test on a saliva stain are shown in Table J. This technique is extremely tedious and time consuming and although adequate to characterize saliva in uncontaminated stains it is not sufficiently sensitive nor sufficiently free from complicating factors to allow identification of saliva in mixed stains. Thus, the starch/iodine reaction is subject to interference by protein and other compounds which sequester iodine, e.g. protein present in blood and choline in semen will compete with unhydrolyzed starch for iodine. Consequently, these body fluids can produce false positive reactions for amylase activity using
2. Detection o f P r o d u c t s o f S t a r c h Hydrolysis The detection of products formed as a result of substrate hydrolysis for the identification of amylase activity in saliva stains is possible if an insoluble amylase/dye complex (Amylose Azure) is used as a substrate. Rosalki (1970) described the use of this compound for the detection of amylase activity. Hydrolysis of this substrate results in the liberation of a soluble blue dye. This substrate has been compared with the starch/iodine method as described in (1) for the detection of amylase activity in saliva stains. The extraction and dilution procedure was precisely the same as described in (1). It has been found, however, that this substrate gives good results with a shorter incubation period at a higher temperature, namely 56° C for 40 min. Substrate hydrolysis after this time results in the release of the blue dye which gives a blue coloration to the incubation medium down to extract dilutions of approximately 1 : 1000. Owing to the greater sensitivity of this substrate in detecting amylase activity, the method devised using this for the detection of amylase activity in saliva stains and saliva/ other body fluid stains is as follows. A i in square piece of stained fabric is incubated in 2 drops of 0·10 per cent suspension of the substrate in buffered saline for
40
Med. Sei. Law (1975) Vol. 1S, No. 1
40 min in a re-suspension this time the visually for
In order to assess this technique for its use in casework investigations a survey was designed in which the operator was unaware of the composition of the stains under test.
56° C incubator, with periodic of the insoluble substrate. After incubation tubes are inspected release of soluble blue dye
Table II: Results or Blind Survey for Amylase Activity on 100 Unknown Stains using Amylose Azure Technique Composition of Stain Saliva—neat Semen Blood Urine Serum Milk Tea Coffee Saliva—boiled Malt extract Yeast extract Potato extract Saliva—1 : 2 Saliva—1 : 4 Saliva—1 ; 8 Saliva—1 :16 Saliva—1 :32 Urine/saliva—1 : 1 Urine/semen-1 ; 1 Blood/saliva—1 : 1 Semen/saliva—1 :1 Serum/saliva—1 : 1 Blood/semen—1 : 1 Total
0
Stains abse&sed to contain Saliva
0 0 0 0 0 0 0 0 0 0 0 0 1 2 0 0 0 0 0 0 0 0 0
0 23 6 4 1 2 1 1 2 2 2 2 0 0 2 2 2 0 3 0 0 0 4
12 0 0 0 0 0 0 0 0 0 0 0 6 4 0 0 0 3 0 6 9 2 0
3
58
42
Score of α-amylase Activity 3 2 1
Number Tested
4
12 23 5 4 1 2 1 1 2 2 2 2 6 4 2 2 2 3 3 6 9 2 4
6 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 4 8 1 0
3 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 1 0 0 1 0 0
3 0 0 0 0 0 0 0 0 0 0 0 2 2 0 0 0 1 0 2 0 1 0
100
21
7
11
Explanation of score of a-amylase activity. 4 = complete hydrolysis of substrate, intense blue supernatant. 3 = Incomplete hydrolysis of substrate, intense blue supernatant. 2 = no visible hydrolysis of substrate, blue supernatant. 1 = no visible hydrolysis of substrate, pale blue supernatant. 0 = no visible hydrolysis of substrate, no coloration of supernatant.
indicative of amylase activity. Unstained cloth controls are treated in a similar fashion at the same time. In our experience there is no need for control procedures other than using a known saliva stain as a positive control. A S S E S S M E N T OF THE TECHNIQUES In addition to being cumbersome and time consuming the starch/iodine detection in our experience is inadequate for the detection of saliva in mixed body fluid stains. The amyloazure method, however, is very simple to perform and is quite sensitive and reliable for establishing the presence of salivary amylase in saliva stains and mixed body fluid stains.
Using the techniques described above in (2) a wide range of stains was tested. As a conse quence of the results obtained using the release of soluble blue dye as a marker, the operator was asked to indicate whether or not saliva was present in these stains. The details of the design of this experiment involving 100 different types of body fluid stains and the results obtained are given in Table II. These show that this technique is positively reliable in that correct interpreta tions for the presence of saliva were made for all of those stains which actually contained saliva. It is possible that with mixed stains which contain less than 1 : 4 saliva this tech nique would fail in practice. However, there
Baxter and Rees: Identification of Saliva
are few instances in which the investigating scientist would be restricted to the use of only i in square of stained material, and using larger amounts of unknown stain material would overcome this. In view of this it must be stressed that failure to detect soluble dye does not necessarily mean that saliva is absent. DISCUSSION During the course of this investigation other possible sources of amylase such as serum, semen, urine and vegetable extracts have been tested. N o amylase activity was detected in any of these test stains other than those containing saliva. Other than blood, semen and vaginal secretion, saliva is the only body fluid which the practising forensic serologist is required to identify on a routine basis. Serological procedures for the identiñcation of blood are long established and well known. The serological identification of semen has been put on a firm practical basis by the combination of immunological and biochemical procedures by Baxter (1973). The possibility of using serological procedures for the identification of salivary amylase has been discussed by Masson et al. (1965). Antisera directed against human saliva are not yet commercially available. The authors recendy obtained a supply of rabbit antisera which had been raised against whole human group A saliva. Our assays of this anti-saliva serum have shown that it is non-specific. Thus its reaction with saliva and semen are completely comparable. We have failed to make this reagent specific by absorption with pooled semen. This antiserum identifies a thermostable antigen which is present as a major component in both saliva and semen and could well be mucoprotein. Attempts to visualize the amylase/antibody complex, after reacting this antiserum with
41
saliva, using the starch/iodine colour reaction as indicator in an analogous fashion to the acid phosphatase precipitin method for seminal acid phosphatase (Baxter, 1973) have also failed. Thus it has not yet been possible to utilize immunological methods for the identification of saliva in body fluid stains. The availability of an immunological method for the identification of saliva could in certain circumstances be of value. The method for detection of products of starch hydrolysis using Amylose Azure is sufficiently sensitive for most practical purposes. Thus this method has been used successfully for the identification of saliva on cigarette ends, postage stamps and envelope flaps. As this represents the smallest amount of saliva which the practising forensic scientist is called upon to investigate, the amylo-azure method is generally adequate for these purposes. The use of this method for the identification of saliva by its amylase activity should always be supplemented by microscopic examination for the presence of buccal epithelia. When used in conjunction these methods represent a reliable procedure for the identification of saliva in body fluid stains.
REFERENCES B a x t e r S. J . (1973) I m m u n o l o g i c a l identification of h u m a n l e m e n . Med. Sei. Law 13, 1 5 5 - 1 6 5 . K i n d S. S. (1964) Methodi of Forensic ScUnce. V o l . 3 . N e w York, Interscience, p . 2 6 7 . Masson P. L., Carbonara A. O . and Heremans J . F. (1965) Studies o n t h e p r o t e i n s of h u m a n saliva. Biochim. Biophys. Acta 107, 4 8 5 - 5 0 0 . Nelson D . F . a n d K i r k P . L . ( 1 % 3 ) T h e identification of saliva. J . Forensic Med. 10, 1 4 - 2 1 . NickoUs L . C . (1956) The Scient^c IiwtsHgation of Crime. L o n d o n , B u t t e r w o r t h , p . 200. Rosalki S. B . (1970) A direct staining t e c h n i q u e for a m y l a s e , isoenzyme d e m o n s t r a t i o n . J. Clin. Pathol. 23, 3 7 3 - 3 7 4 . W h i t e h e a d P . H . (1974) p e r s o n a l c o m m u n i c a t i o n .
37
The Identification of Saliva in Stains in Forensic Casework S. J . BAXTER. BSC. PhD B. REES. BSC. PhD Home Counties Forensic Science Aldermaston, Reading
Laboratory,
INTRODUCTION The evidential value of saliva in stains encountered in crime investigation can be of considerable importance. Whether the stains are a result of expectoration or a consequence of some sexual or violent act resulting in saliva being mixed with other body fluids, the practising forensic scientist is faced with two fundamental problems: (1) localization of the stain, and (2) identification of the stain. In practice these two aspects arc often closely interlinked. Visualization of saliva stains is problematical. Unlike blood which has its own internal marker, i.e. haemo globin, saliva h u no luch readily visible constituent. In this respect it is similar to semen. The introduction of the acid phos phatase test for the location of semen stains has, however, overcome these problems. Kind (1964) has reviewed the acid phos phatase test for the identification of semen. As yet no test which compares with the acid phosphatase test for the localization of semen has been devised for saliva, although the use of Procion Red Μ χ 2B amylopectin for the visualization of amylase activity promises to parallel the acid phosphatase test in this respect (Whitehead, 1974). The literature on the identification of saliva stains is not extensive. The use of the amylase activity of saliva for its identification has been described by Nickolls (1956). Nelson and Kirk (1963) dealt more compre hensively with the possible markers for the identification of saliva. Human saliva is a very complex fluid but there appear to be 4 components which can be considered to offer
possible parameters for its identification. These are: 1. Enzymes (α-amylase, alkaline phos phatase) or proteins with specific activity such as lysozyme. 2. Non-cnzymic proteins—mucoproteins. 3. Buccal epithelial cells. 4. Inorganic components (nitrite and thiocyanate). Although there would appear to be few restrictions on the number of possible markers .for the identification of saliva, in practice there are considerable problems. As a consequence the identification of saliva stains and saliva in mixed stains has mainly relied on the amylase activity of saliva as detected by starch hydrolysis allied with microscopy for buccal epithelial cells. Other constituents which offer possible means of characterizing saliva are such that for practical purposes they have not found wide usage. Thus, the value of alkaline phosphatase, nitrite and thiocyanate in establishing the salivary origin of suspect material has been fully described by Nelson and Kirk (1963) and the authors are in agreement with their evaluation. Similar criticisms can be used against the use of lysozyme as a possible means for the identi fication of saliva. Thus, although this protein can be readily identified by immunological procedures, its wide occurrence in other body fluids such as semen and lacrimal fluid renders this of little value. T h e value of amylase as a means for detecting saliva is still of prime importance and will be discussed later.
38
Med. Sei. Law (1975) Vol. 15, No. 1
This paper reports work carried out in this laboratory which is concerned with bio chemical methods for the identification of saliva in stain and mixed body fluid stains.
buffer in the upper compartment of the extraction device illustrated in Fig. 1 . After this period the extraction device is centrifuged and the stain extract can then be removed from the lower compartment. This
MATERIALS Amylose Azure was obtained from Calbiochem Limited, 10 Wyndham Place, London. Barbitone buffered saline (sterile) p H 7-2 5x concentrated was obtained from Wellcome Reagents, Beckenham, Kent. Soluble Starch Analar grade was obtained from B D H Chemicals Limited, Poole, Dorset. METHODS The only satisfactory procedure for the identification of saliva in stains, of which the authors are aware, relies upon the high amylase activity of saliva. Thus, there are two methods whereby starch hydrolysis can be detected. The first of these relies upon the detection of unhydrolyzed starch, the second depends on the detection of products formed as a result of substrate hydrolysis. These two methods are dealt with separately. 1 . Detection o f Unhydrolyzed Starch Nickolls (1956) and Nelson and Kirk (1963) described methods which utilize the detection of unhydrolyzed starch for the identification of salivary amylase. The routine procedure used in this laboratory for the identification of salivary amylase in stains is a refinement of these techniques. This technique involves serial dilution of the material under investigation prior to incubation with soluble starch. In this method doubling dilutions up to 1 : 256 are prepared from salivary stain extracts. The details of the procedure are as follows. After localization of the suspected areas of saliva staining as much of the stained area as can be afforded, without prejudicing other test procedures, is removed from the garment. In the case of neat, uncontaminated saliva stairts ^ sq in of stained material is sufficient. Proportionately more is required in cases with smeared saliva stains or mixtures with other body fluids. The stained area after removal is extracted for J hr in 7 drops of
Fig. J: Extraction device. A, upper polystyrene tube 36 mm χ 6 mm; B, transparent adhesive tape snap-joint; C, small hole; D, lower polystyrene tube 36 mm χ β mm.
normally results in approximately 6 drops of stain extract being recovered. These are then treated as follows. Three drops of the extract are removed and heated in a sealed glass tube at 100° C for 10 min. T w o drops of the initial extract plus 2 drops of the boiled extract are then separately and serially diluted in glass tubes to dilutions of 1 : 256 using the same buffer as used for the stain extraction. T o each of these dilutions is added 1 drop of 1 per cent soluble starch. All tubes are then incubated at 37° C for 1 hr after which time 1 drop of iodine solution is added. Any
Baxter and Rees: Identification of Saliva
unhydrolyzed starch will be detected by the characteristic intense blue starch/iodine reaction. It is essential to treat a control area of cloth in precisely similar fashion.
39
the techniques described. Therefore, the presence of saliva in mixed stains with these body fluids cannot be satisfactorily established using this procedure.
Table I: Reactions of Diluted Saliva Stain Extracts with 1 per cent Soluble Starch using Starch/Iodine Colour Reaction as Indicator of Degree of Starch Hydrolysis
Extract Dilutions
Colour Reaction with Iodine after 1 hr Incubation at 37°C with 1 drop 1 per cent Starch Solution Neat 1 : 2 1 :4 1:8 1 :16 1 :32 1 :64 1 :128 1 :256
Extracted saliva stain
Untreated extract Boiled extract
—
Extracted control area
Untreated extract Boiled extract
— —
+ + +
+
+
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ = deep blue colour; + = faint blue colour; — = no blue colour.
Using this method an indication of the amylase activity in the stain extract together with its serial dilutions is obtained. In normal saliva stains complete starch hydrolysis occurs in the diluted extract at dilutions of 1 : 64 and sometimes to 1 : 128. In the boiled extract starch hydrolysis should not occur at all as the enzyme will have been heat inactivated. It does occur, however, that non-speciñc contaminating materials in the cloth can prevent the starch/iodine colour reaction so giving rise to false positive reactions unless adequate control procedures are taken. These false positive reactions rarely extend as far as the 1 : 8 dilution, however, indicating that this is caused by some agent in the cloth whose action is not enzymic. Typical results for such a test on a saliva stain are shown in Table J. This technique is extremely tedious and time consuming and although adequate to characterize saliva in uncontaminated stains it is not sufficiently sensitive nor sufficiently free from complicating factors to allow identification of saliva in mixed stains. Thus, the starch/iodine reaction is subject to interference by protein and other compounds which sequester iodine, e.g. protein present in blood and choline in semen will compete with unhydrolyzed starch for iodine. Consequently, these body fluids can produce false positive reactions for amylase activity using
2. Detection o f P r o d u c t s o f S t a r c h Hydrolysis The detection of products formed as a result of substrate hydrolysis for the identification of amylase activity in saliva stains is possible if an insoluble amylase/dye complex (Amylose Azure) is used as a substrate. Rosalki (1970) described the use of this compound for the detection of amylase activity. Hydrolysis of this substrate results in the liberation of a soluble blue dye. This substrate has been compared with the starch/iodine method as described in (1) for the detection of amylase activity in saliva stains. The extraction and dilution procedure was precisely the same as described in (1). It has been found, however, that this substrate gives good results with a shorter incubation period at a higher temperature, namely 56° C for 40 min. Substrate hydrolysis after this time results in the release of the blue dye which gives a blue coloration to the incubation medium down to extract dilutions of approximately 1 : 1000. Owing to the greater sensitivity of this substrate in detecting amylase activity, the method devised using this for the detection of amylase activity in saliva stains and saliva/ other body fluid stains is as follows. A i in square piece of stained fabric is incubated in 2 drops of 0·10 per cent suspension of the substrate in buffered saline for
40
Med. Sei. Law (1975) Vol. 1S, No. 1
40 min in a re-suspension this time the visually for
In order to assess this technique for its use in casework investigations a survey was designed in which the operator was unaware of the composition of the stains under test.
56° C incubator, with periodic of the insoluble substrate. After incubation tubes are inspected release of soluble blue dye
Table II: Results or Blind Survey for Amylase Activity on 100 Unknown Stains using Amylose Azure Technique Composition of Stain Saliva—neat Semen Blood Urine Serum Milk Tea Coffee Saliva—boiled Malt extract Yeast extract Potato extract Saliva—1 : 2 Saliva—1 : 4 Saliva—1 ; 8 Saliva—1 :16 Saliva—1 :32 Urine/saliva—1 : 1 Urine/semen-1 ; 1 Blood/saliva—1 : 1 Semen/saliva—1 :1 Serum/saliva—1 : 1 Blood/semen—1 : 1 Total
0
Stains abse&sed to contain Saliva
0 0 0 0 0 0 0 0 0 0 0 0 1 2 0 0 0 0 0 0 0 0 0
0 23 6 4 1 2 1 1 2 2 2 2 0 0 2 2 2 0 3 0 0 0 4
12 0 0 0 0 0 0 0 0 0 0 0 6 4 0 0 0 3 0 6 9 2 0
3
58
42
Score of α-amylase Activity 3 2 1
Number Tested
4
12 23 5 4 1 2 1 1 2 2 2 2 6 4 2 2 2 3 3 6 9 2 4
6 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 4 8 1 0
3 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 1 0 0 1 0 0
3 0 0 0 0 0 0 0 0 0 0 0 2 2 0 0 0 1 0 2 0 1 0
100
21
7
11
Explanation of score of a-amylase activity. 4 = complete hydrolysis of substrate, intense blue supernatant. 3 = Incomplete hydrolysis of substrate, intense blue supernatant. 2 = no visible hydrolysis of substrate, blue supernatant. 1 = no visible hydrolysis of substrate, pale blue supernatant. 0 = no visible hydrolysis of substrate, no coloration of supernatant.
indicative of amylase activity. Unstained cloth controls are treated in a similar fashion at the same time. In our experience there is no need for control procedures other than using a known saliva stain as a positive control. A S S E S S M E N T OF THE TECHNIQUES In addition to being cumbersome and time consuming the starch/iodine detection in our experience is inadequate for the detection of saliva in mixed body fluid stains. The amyloazure method, however, is very simple to perform and is quite sensitive and reliable for establishing the presence of salivary amylase in saliva stains and mixed body fluid stains.
Using the techniques described above in (2) a wide range of stains was tested. As a conse quence of the results obtained using the release of soluble blue dye as a marker, the operator was asked to indicate whether or not saliva was present in these stains. The details of the design of this experiment involving 100 different types of body fluid stains and the results obtained are given in Table II. These show that this technique is positively reliable in that correct interpreta tions for the presence of saliva were made for all of those stains which actually contained saliva. It is possible that with mixed stains which contain less than 1 : 4 saliva this tech nique would fail in practice. However, there
Baxter and Rees: Identification of Saliva
are few instances in which the investigating scientist would be restricted to the use of only i in square of stained material, and using larger amounts of unknown stain material would overcome this. In view of this it must be stressed that failure to detect soluble dye does not necessarily mean that saliva is absent. DISCUSSION During the course of this investigation other possible sources of amylase such as serum, semen, urine and vegetable extracts have been tested. N o amylase activity was detected in any of these test stains other than those containing saliva. Other than blood, semen and vaginal secretion, saliva is the only body fluid which the practising forensic serologist is required to identify on a routine basis. Serological procedures for the identiñcation of blood are long established and well known. The serological identification of semen has been put on a firm practical basis by the combination of immunological and biochemical procedures by Baxter (1973). The possibility of using serological procedures for the identification of salivary amylase has been discussed by Masson et al. (1965). Antisera directed against human saliva are not yet commercially available. The authors recendy obtained a supply of rabbit antisera which had been raised against whole human group A saliva. Our assays of this anti-saliva serum have shown that it is non-specific. Thus its reaction with saliva and semen are completely comparable. We have failed to make this reagent specific by absorption with pooled semen. This antiserum identifies a thermostable antigen which is present as a major component in both saliva and semen and could well be mucoprotein. Attempts to visualize the amylase/antibody complex, after reacting this antiserum with
41
saliva, using the starch/iodine colour reaction as indicator in an analogous fashion to the acid phosphatase precipitin method for seminal acid phosphatase (Baxter, 1973) have also failed. Thus it has not yet been possible to utilize immunological methods for the identification of saliva in body fluid stains. The availability of an immunological method for the identification of saliva could in certain circumstances be of value. The method for detection of products of starch hydrolysis using Amylose Azure is sufficiently sensitive for most practical purposes. Thus this method has been used successfully for the identification of saliva on cigarette ends, postage stamps and envelope flaps. As this represents the smallest amount of saliva which the practising forensic scientist is called upon to investigate, the amylo-azure method is generally adequate for these purposes. The use of this method for the identification of saliva by its amylase activity should always be supplemented by microscopic examination for the presence of buccal epithelia. When used in conjunction these methods represent a reliable procedure for the identification of saliva in body fluid stains.
REFERENCES B a x t e r S. J . (1973) I m m u n o l o g i c a l identification of h u m a n l e m e n . Med. Sei. Law 13, 1 5 5 - 1 6 5 . K i n d S. S. (1964) Methodi of Forensic ScUnce. V o l . 3 . N e w York, Interscience, p . 2 6 7 . Masson P. L., Carbonara A. O . and Heremans J . F. (1965) Studies o n t h e p r o t e i n s of h u m a n saliva. Biochim. Biophys. Acta 107, 4 8 5 - 5 0 0 . Nelson D . F . a n d K i r k P . L . ( 1 % 3 ) T h e identification of saliva. J . Forensic Med. 10, 1 4 - 2 1 . NickoUs L . C . (1956) The Scient^c IiwtsHgation of Crime. L o n d o n , B u t t e r w o r t h , p . 200. Rosalki S. B . (1970) A direct staining t e c h n i q u e for a m y l a s e , isoenzyme d e m o n s t r a t i o n . J. Clin. Pathol. 23, 3 7 3 - 3 7 4 . W h i t e h e a d P . H . (1974) p e r s o n a l c o m m u n i c a t i o n .
