Evaluation of amylase testing as a tool for saliva screening of crime scene trace swabs

Amylase testing has been used as a presumptive test for crime scene saliva for over three decades, mainly to locate saliva stains on surfaces. We have developed a saliva screening application for crime scene trace swabs, utilising an amylase sensitive paper (Phadebas^® Forensic Press test). Positive results were obtained for all tested dried saliva stains (0.5–32 μL) with high or intermediate amylase activity (840 and 290 kU/L). Results were typically obtained within 5 min, and all samples that produced DNA profiles were positive. However, salivary amylase activities, as well as DNA concentrations, vary significantly between individuals. We show that there is no correlation between amylase activity and amount of DNA in fresh saliva. Even so, a positive amylase result indicates presence of saliva, and thereby presence of DNA. Amylase testing may be useful for screening in investigations where the number of DNA analyses is limited due to cost, e.g., in volume crime.     

Comparison of Catalytic and Immunological Amylase Tests for Identifying of Saliva from Degraded Samples

The stability of salivary α‐amylase is a critical factor in both catalytic and immunological method‐based forensic saliva identification. This study aimed to assess the sensitivity of catalytic and immunological tests on degraded saliva samples. Degraded saliva stains were prepared by microbial decomposition using humid soil. Salivary α‐amylase activity was catalytically detected both qualitatively and quantitatively using the Phadebas^® amylase test. As immunological methods, we conducted qualitative and quantitative tests using the RSID?‐saliva test and ELISA, respectively. Salivary α‐amylase activity of degraded samples (incubated at 37°C for 12 h) was significantly lower than that of controls in the quantitative tests. All the degraded samples obtained by the humid soil produced negative results in the Phadebas^® tests, but showed positive results in the RSID?‐saliva test and ELISA. These results suggest that immunological tests are effective for testing degraded saliva samples that have lost their enzymatic activity.     

Applicability of two commercially available kits for forensic identification of saliva stains

The RSID-saliva test and the SALIgAE-saliva test are two recently developed forensic saliva detection kits. In this study, we compared the sensitivity and the specificity of the two test kits with the Phadebas amylase test by analyzing amylases from various sources including human, animals, plants, and micro-organism. The data demonstrate that the RSID-saliva test and the SALIgAE-saliva test offer higher sensitivity and specificity for the detection of saliva than the Phadebas amylase test. The detection limits of the RSID-saliva test, the SALIgAE-saliva test, and the Phadebas amylase test equate to 10, 4, and 1000 nL, respectively for human saliva. The RSID-saliva test and the SALIgAE-saliva test were further evaluated by analyzing semen, vaginal secretion, breast milk, blood, urine, sweat, and feces. The results of the two tests are in good agreement. The two tests reacted with urine, breast milk, and feces, but not with semen, vaginal secretion, blood, and sweat.     

An ELISA method for the identification of salivary amylase

An ELISA method for the detection of salivary amylase in dried stains using a monoclonal anti-human salivary amylase antibody was developed. Studies demonstrated the assay to be sensitive down to 0.0002 Sigma units and showed a linear response between absorbance and salivary amylase activity between 0.002 and 0.2 units. The assay showed no cross reactivity with either commercially purchased human pancreatic or bacterial amylase. Sample studies utilizing swabs from several human body fluids showed that 100% of all saliva containing swabs (sixteen of sixteen) and 13% of non-saliva human body fluid swabs (eight of sixty-three) showed a net absorbance with the method. Of these eight non-saliva swabs yielding a net absorbance, none exceeded a salivary amylase activity of 0.003 units. In contrast, only three of the sixteen saliva-containing swabs (swabs produced from saliva diluted 1:5, 1:6, and 1:10, respectively) showed an activity below 0.2 units. Of these swabs, the 1:100 dilution showed the lowest activity (0.048). This value is still more than ten times that of the non-saliva containing swab with the highest activity.     

A rapid method to detect dried saliva stains swabbed from human skin using fluorescence spectroscopy

Saliva on skin is important in forensic trace evidence. If areas where saliva is present can be outlined, this may lead to DNA analysis and identification. This study describes a rapid and non-destructive method to detect dried saliva on the surface of the skin by fluorescence spectroscopy. Eighty-two volunteers deposited samples of their own saliva on the skin of their ventral forearm. A control sample of water was deposited at three different sites on the contralateral arm. Saliva and water control were then allowed to air-dry. Swab samples were taken from dried saliva and control sites and were dissolved in 0.1M KCl solution. Emission spectra were obtained from the solution and were characterized by a principal maximum at 345-355nm with excitation at 282nm. The fluorescence emission intensity was greater than background readings obtained from the control swab site in 80 of 82 volunteers (approximately 97.6%). The fluorescence profile of saliva samples were similar to those obtained from aqueous samples of pure amylase and tryptophan, an endogenous fluorophore in alpha-amylase. The presence of an emission peak at 345-355nm with excitation at 282nm could provide a strong presumptive indication of saliva deposition.     

Amylase levels in semen and saliva stains

Amylase levels were determined for 148 semen samples and 20 saliva samples as well as for their corresponding stains. The effect of aging on the detectability of amylase activity in these stains was also investigated. The Phadebas amylase test was used for the quantitative assay of amylase. High levels of amylase in fluid saliva resulted in high levels being detected in saliva stains. Lower levels present in most seminal fluids produce little or no detectable amounts of amylase in stains. Interpretations are made as to the possible sources of amylase activity found in stains from laboratory casework based on both the amylase concentration and the elapsed time between collection and analysis. The evidential value of the presence or absence of amylase activity in casework stains is also discussed.     

False‐Positive Results with Amylase Testing of Citrus Fruits

In a case of robbery in which the criminals passed through the garden adorned with calamondin trees (Citrus madurensis), the investigators found in the grass six calamondin fruits, some undamaged, while others apparently bitten. The fruits were collected and sent to the laboratory for DNA analysis to verify the presence of saliva and robbers' DNA profile. A specific immunochromatographic strip test for saliva confirmed the presence of human salivary α‐amylase, but similar positive results were also observed for intact calamondin and other citrus fruits. Further analysis with a specific automated amylase test confirmed the absence of amylase activity. DNA quantification and typing using a specific forensic kit revealed no human DNA presence in any fruits. This case report demonstrates for the first time the occurrence of false positives when human saliva is sought on citrus fruits.     

The rapid determination of the ABO group from body fluids (or stains) by dot enzyme-linked immunosorbent assay (dot-ELISA) using enzyme-labeled monoclonal antibodies

Using ABH enzyme-labeled monoclonal antibodies, the authors could rapidly detect the ABO group from body fluids and body fluid stains by the dot enzyme-linked immunosorbent assay (dot-ELISA). In this test, the antigen was immobilized on nitrocellulose paper; the entire piece of paper was coated with an appropriate dilution of enzyme-labeled McAb directly against the antigen of interest; and, finally, 3,3'-diaminobenzidine (DAB) substrate solution was added. The site of a positive reaction is clearly visible as a brown spot. We analyzed 521 samples and got satisfactory results. We also analyzed 99 practical case samples by this method and achieved the same results as those obtained by other researchers using other methods. This method is accurate, simple, direct, rapid, and sensitive; it also produces easily observed results, requires no equipment, and can be completed in 30 min. The test proved to be clearly more sensitive for the detection of the ABO blood group in secretor saliva than the conventional hemagglutination inhibition test. Also saliva diluted 10(-4) to 10(-5) and the ABO group of nonsecretor saliva and urine could be easily detected by this method.     

Assessing the background levels of body fluids on hands

Forensic scientists are often asked to assist the court by evaluating the significance of finding body fluids on the hands of an individual; however, there is an absence of published data regarding the background levels of body fluids on hands. Whilst the scientist can use casework experience to inform the courts on the significance of the results, it would be advantageous to have data which could assist with this interpretation. This study was designed to ascertain the background levels of blood, semen, saliva, hairs/fibres and staining/debris on hands in the general population by sampling from delegates attending a scientific conference.The findings suggest that approximately one third of the population would be expected to have hairs or fibres on their hands and that females are more likely to have visible staining on their hands than males. Presumptive tests for blood and semen yielded negative results in all samples; however, almost 2 % of the samples were found to contain a very low number of sperm heads. In contrast, the majority of samples tested positive for the presence of saliva using the presumptive Phadebas® amylase test. The data supports the caution applied by forensic practitioners when evaluating the presence of saliva detected using the presumptive Phadebas® amylase test based on the lack of specificity and indicates that the RSID™-Saliva test would be more suitable to use.     

Comparison of modern techniques for saliva screening

Saliva stains present a unique challenge in the forensic setting, often challenging the analyst to weigh the value of presumptive indication of the fluid versus the potential for DNA analysis to yield identification information. There are many situations in which determining the presence of a body fluid is probative and further corroborates DNA evidence. That said, even a minute portion of sample consumed by a screening test could mean the difference between a full, partial, or null profile obtained through DNA analysis. The basis of presumptive testing or screening of saliva has historically been based on the presence of amylase, a component found in relatively high concentrations in human saliva versus other body fluids and substances. Though the current available methods for the screening of saliva in a forensic application have grown in number, the popularity of these methods seemingly has not. This study attempts to identify a specific and sensitive saliva screening test by comparing three modern techniques--the recently released SALIgAE, Phadebas, and starch-iodine mini-centrifuge test--on the basis of sensitivity, specificity, mixtures, and simulated casework samples while also considering sample consumption. The Phadebas method for presumptive saliva testing detected dilutions of neat saliva down to 1:200 versus considerably less sensitive results with SALIgAE and the starch-iodine mini-centrifuge test. Utilizing a screening test with a high degree of sensitivity, such as Phadebas, allows an analyst to gain a maximum amount of information in the form of body fluid indication and DNA results because of the consumption of a small portion of sample.     

检测体液中氰含量的荧光光度法研究

本文报道了应用取代反应检测体液中氰含量的荧光光度法。探讨了检测的具体方法和有关条件对检测的影响。检测了正常人血、尿液和唾液中氰含量,并检测了小鼠氰化物染毒死亡后血中氰含量。研究表明,本法不但可检测体液中致死量的氰化物含量,亦可检测体液中正常氰含量。     

Determination of phenotypes of salivary amylase in liquid saliva and saliva stains

An isoelectric focusing method is described for typing salivary amylase in liquid saliva and saliva stains. The estimated gene frequencies in a British population, calculated on the basis of three alleles operating at a single locus, were Amy 1, 0.909; Amy 2, 0.065; Amy 3, 0.026. This system may be useful in forensic investigations.     

Identification of saliva stains by determination of the specific activity of amylase.

The specific activity (enzyme activity/protein concentration) of amylase was determined for the identification of saliva stains. The specific activity of amylase in saliva stains rapidly decreased during the first hour but, from 1 to 28 days, this decrease was much less when the stains were kept at room temperature. Stains of various human biological materials, breast milk, nasal secretion, meconium and vaginal secretion showed comparatively high amylase activity, but the saliva stains could be differentiated by their high specific activity of amylase, over 2 I.U./mg. When saliva stains were contaminated with blood or vaginal secretions at various ratios, the specific activity of amylase decreased with increase in the ratio of contaminant, especially when the contaminant was blood. However, the specific activity of amylase was still higher than 2 I.U./mg even after one fifth volume of blood was added or after five volumes of the extract of the stains of vaginal secretions were added.     

The examination of vaginally inserted plastic tampon applicators for genetic markers and evidence of prior sexual intercourse

Vaginally inserted plastic tampon applicators were obtained from 42 female volunteers. The applicators were examined for the presence of ABH blood group substances, phosphoglucomutase (PGM), amylase, acid phosphatase, P30, and intact spermatozoa. Each applicator was accompanied by a control blood sample, a saliva specimen, a brief sexual and menstrual history, and method of birth control of the donor. Eight of the male sexual partners of the donors submitted blood and saliva samples. One male sexual partner submitted only a saliva sample. ABH blood group substances corresponding to the donor were recovered from 36 of the 42 applicators. The remaining 6 applicators revealed a combination of the donor's and sexual partner's ABH substances. The female's PGM type was recovered from 34 of the applicators. The remaining 8 applicators failed to show PGM activity. Of the applicators, 15 indicated evidence of prior sexual intercourse by the detection of ABH substances not consistent with the applicator donor (6 samples), high levels of acid phosphatase (11 samples), or recovery of spermatozoa (8 samples) or some combination of these. All applicator samples failed to show the presence of either P30 activity or PGM factors foreign to the female.     

A new method for locating saliva stains-spotty paper for spotting spit

A convenient method of locating saliva stains has been devised. Filter paper covered with blue coloured starch fragments is laid on the material to be tested. Salivary amylase hydrolyses the starch on the areas of paper corresponding to saliva stains, which are then delineated as clear zones whereas negative areas remain speckled with blue particles.     

A sensitive and simple assay of saliva on stamps

Identification of saliva on stamps or envelope flaps remains yet a not widely studied problem. In most forensic laboratories it is seldom carried out, but this fact does not reduce the importance of the assay. Most authors consider amylase a sufficiently specific marker of the presence of saliva; really, the only other human body fluid that contains high amounts of this enzyme is the pancreatic juice (and therefore feces). Here we present a simple and sensitive assay for the determination of alpha-amylase that uses a commercially available and well-known substrate. It is hydrolyzed by amylase with the production of soluble blue fragments, that can be measured by photometry, obtaining objective results. The presented assay identifies 1 X 10(-6) diluted saliva or that present on 0.5 mg of a stamp; 16-year-old samples can also be identified. Intra-assay and day-to-day CV resulted in 10.8% and 13.7%, respectively. Owing to the high sensitivity of the test, handling samples or reagents can introduce contamination with saliva traces, giving false-positive results. Addition of EDTA 0.1 mol/l to the incubation mixture, lowering the sensitivity to 1 X 10(-3) diluted saliva, overcomes this problem.     

Examination of the correlation of groupings in blood and semen

The grouping of blood/saliva samples from a male so as to predict his semen groups is only justified if there is a strict correlation between the groupings in these body fluids. This correlation has been examined in the ABO, phosphoglucomutase (PGM1) and glyoxalase I (GLO) grouping systems in blood and semen samples collected from more than 250 individuals. Though no results proved inconsistent with this correlation, a number of semen gave inconclusive grouping results. Reasons for this are discussed as well as the relevance of the results to semen stain analysis. Semen amylase activities are also reported.     

Detection of saliva traces using test strips

The test strip Rapignost-Amylase (Behring) for the rapid determination of alpha-amylase in the urine is also suitable for the determination of salivary amylase in stains stored up to 6 weeks at room temperature. The stains are extracted with physiological saline (extraction time 30 min), then the application zone of the strip is wetted with the extract. Positive amylase-reaction is recognised as a reddish-violet colouration of the reaction zone. Biological stains with low amylase concentrations (urine semen, vaginal secretion, mucus) react amylase negative. The method is uncomplicated and can be completed within 30 min. The test strips are easily available and stable during storage. Therefore the determination of saliva with test strips should be preferred to the clinical methods if the storage times of the stain are not longer than 4-6 weeks. It is a suitable procedure to determine salivary stains for use in forensic biology.     

Diagnostics of the AB group of AB0(H) system in saliva traces

On the basis of the mixed agglutination reaction of the saliva spots, obtained from persons with groups A, B, and AB (extractors) as well as mixtures of saliva obtained from the above subjects in various combinations, were experimentally studied. It was shown to be possible to diagnose the AB group both in the "pure" saliva traces and as an admixture of the AB group saliva to the groups A and B saliva obtained by using the anti-A and anti-B hetero-immune gemagglutinating sera. Our results confirm the previously published opinion on that antigens A and B of the ABse group saliva are positioned in the cis-acting mode, i.e. they are located on one molecule of surface structure of the epithelial (buccal) cell.     

Glycogenated squamous epithelial cells as a marker of foreign body penetration in sexual assault

Nonconsensual insertion of a foreign object into the vagina, anus, or mouth in some judicial jurisdictions is synonymous with rape, and elsewhere may constitute some degree of sexual assault or battery. Few techniques, however, are available to assist the criminalist in determining whether an object has been criminally inserted. Glycogenated epithelial cells have been used as a marker for vaginal epithelium, and as such, may indicate vaginal insertion if recovered from an object. This hypothesis was tested by studying orally and vaginally inserted objects from 42 volunteers and 20 rectally inserted objects recovered from cadavers. Glycogen positivity was assayed from smears of object swabbings stained with the periodic acid-Schiff (PAS) technique. More than 75 glycogen positive cells were recovered from 39 of 42 vaginally inserted objects. Glycogenated cells were recovered from 8 of 20 rectally inserted objects (5 with more than 100 positive cells). Of 42 orally inserted objects, 32 also contained glycogen positive cells, but none with more than 28 positive cells. No glycogen positive cells were seen in skin exposed but not inserted objects. Large numbers of glycogen cells were seen in dried saliva drops. Amylase activity was not seen on 5 of 20 orally inserted shields, and thus the possibility of noninsertional saliva contamination could not be ruled out with shields yielding only small numbers of positive cells. Recovery of large numbers of glycogenated cells from foreign objects is strongly suggestive of either vaginal or anal insertion assuming amylase negativity. Glycogen positive cells are not seen secondary to glabrous skin exposure.