Chemical enhancement of footwear impressions in blood on fabric — Part 2: Peroxidase reagents

This study investigates the optimisation of peroxidase based enhancement techniques for footwear impressions made in blood on various fabric surfaces. Four different haem reagents: leuco crystal violet (LCV), leuco malachite green (LMG), fluorescein and luminol were used to enhance the blood contaminated impressions.The enhancement techniques in this study were used successfully to enhance the impressions in blood on light coloured surfaces, however, only fluorescent and/or chemiluminescent techniques allowed visualisation on dark coloured fabrics, denim and leather. Luminol was the only technique to enhance footwear impressions made in blood on all the fabrics investigated in this study.     

The presumptive reagent fluorescein for detection of dilute bloodstains and subsequent STR typing of recovered DNA

A presumptive reagent for dilute blood detection other than luminol is fluorescein. The sensitivity of fluorescein approaches the sensitivity of detection levels of luminol. The fluorescein detection method offers the advantages of working in a lighted environment, and the reaction persists longer than luminol. A series of diluted bloodstains, ranging from neat to 1:1,000,000, was placed on a variety of substrates. Three sets were made per substrate. One set was exposed to fluorescein, one set was exposed to luminol, and one set served as an uncontaminated control. The fluorescein signal persisted longer than luminol. However, background staining for fluorescein was observed on some substrates within 30 s to 1 min, and no background staining was observed for luminol. Stains on non-absorbent surfaces were detectable at 1:100,000 dilutions, and stains on absorbent surfaces were detectable usually at no more than 1:100. The sensitivity of detection of fluorescein was comparable to that of luminol in this study. In all cases, where sufficient DNA was recovered, typeable results at all 13 core CODIS STR loci were obtained from treated bloodstains and controls. The results from STR typing indicate that there was no evidence of DNA degradation.     

The analysis of biological samples from crime scene for a future human DNA profile confrontation. Effects of presumptive test reagents on the ability to obtain STR profiles for human identification

Because of the increase of evidence of blood stains, that have been washed or cleaned in an attempt to mask the analysis of DNA profiles, there is also an increase in the use of presumptive tests on samples sent to laboratories. Some of the presumptive tests, used to identify blood and semen stains, could potentially affect the recovery of high molecular weight DNA from the samples, or extinguish them, especially those already present in small quantities. After the presumptive tests, often these samples are discarded. This study aimed to examine the possibility of obtaining a DNA profile from samples submitted for presumptive testing and cleaned with bleaches with and without chlorine. Two different protocols were conducted: (a) A unique sample of human blood in natura (5 μL), already typed through the DNA techniques with the genetic profile previously known (control), was distributed onto cotton fabrics and dried at room temperature. Four samples of fabric were macerated in saline solution and Coombs serum and then stored for three months (room temperature and freezer −20 °C). (b) Another sample of human blood, type A, in natura, already typed through the techniques of DNA (control) was used. Aliquots of 200 μL were distributed in: cotton, denim and synthetic fabric. The samples were dried at room temperature for 24 h. The blood stains in those fabrics (cotton, denim and synthetic) were then divided into three groups: unwashed, cleaned with chlorine bleach and cleaned with chlorine bleach and soap powder. The samples were again dried at room temperature for 24 h, before the use of luminol. The DNA were extracted with Chelex 100 and amplified with the Identifiler Kit (Applied Biosystems). The blood stains exposed to saline and Coombs serum had DNA profiles consistent with untreated samples (controls). This result shows that the experts should keep and store the samples treated with saline and Coombs serum for future DNA confrontation when necessary. Also discussed in this paper the pattern of blood stains after washing with bleaching solutions, as well as the quantity of DNA obtained from these samples.     

Improving Luminol Blood Detection in Forensics

The aim of this study was to develop chemical improvements to the original Weber protocol, in order to increase the intensity and time length of light emission and to eliminate false‐positive reactions. The intensity and duration of light were measured on serial blood dilutions using a plate reader chemiluminometer. Blood stains of various concentrations were impregnated in pure cellulose, dried, and luminol solution was added with/without the potential enhancers. An in silico study was also conducted, aiming to demonstrate the enhancing mechanism of hemoglobin denaturation using 8 M urea. The luminol blood detection test revealed important improvements after urea pretreatment or in the presence of monochloro‐triazinyl‐β‐cyclodextrin. This approach also eliminated the false‐positive reaction from sodium hypochlorite. These improvements could provide a higher sensitivity under particular circumstances such as old or washed blood stains, leading to a better localization for further DNA typing and higher quality photographic analysis.     

The use of Polilight in the detection of seminal fluid, saliva, and bloodstains and comparison with conventional chemical-based screening tests

Biological stains can be difficult to detect at crime scenes or on items recovered from crime scenes. The use of a versatile light source may assist in their detection. The ability of Polilight to locate potential semen, saliva, and blood stains on a range of substrates and at different dilutions was tested. We also tested the use of Polilight in comparison with conventional chemical-based presumptive screening tests such as acid phosphatase (AP), Phadebas, and luminol, often used in casework for detecting potential semen, saliva, and blood stains, respectively. The Polilight was able to locate stains that were not apparent to the naked eye. The color of the material on which a stain is deposited can have an effect on the detectibility of the stain. The Polilight was found to be comparable with the AP and Phadebas tests in terms of its sensitivity. In a comparative study between the AP test and Polilight on 40 casework exhibits, one false-negative result was observed when using the Polilight. On a series of mock casework exhibits it was determined that the Polilight can be used successfully to locate saliva stains for DNA analysis. The sensitivity of luminol for detecting potential bloodstains was greater than that of Polilight; however the Polilight has particular application in instances where a bloodstain may have been concealed with paint. Overall, the Polilight is a relatively safe, simple, noninvasive, and nondestructive technique suitable for use in forensic casework.     

Fingerprint enhancement revisited and the effects of blood enhancement chemicals on subsequent profiler Plus fluorescent short tandem repeat DNA analysis of fresh and aged bloody fingerprints

This study was aimed at determining the effect of seven blood enhancement reagents on the subsequent Profiler Plus fluorescent STR DNA analysis of fresh or aged bloody fingerprints deposited on various porous and nonporous surfaces. Amido Black, Crowle's Double Stain. 1,8-diazafluoren-9-one (DFO), Hungarian Red, leucomalachite green, luminol and ninhydrin were tested on linoleum, glass, metal, wood (pine, painted white), clothing (85% polyester/15% cotton, 65% polyester/35% cotton, and blue denim) and paper (Scott 2-ply and Xerox-grade). Preliminary experiments were designed to determine the optimal blood dilutions to use to ensure a DNA typing result following chemical enhancement. A 1:200 blood dilution deposited on linoleum and enhanced with Crowle's Double Stain generated enough DNA for one to two rounds of Profiler Plus PCR amplification. A comparative study of the DNA yields before and after treatment indicated that the quantity of DNA recovered from bloody fingerprints following enhancement was reduced by a factor of 2 to 12. Such a reduction in the DNA yields could potentially compromise DNA typing analysis in the case of small stains. The blood enhancement chemicals selected were also evaluated for their capability to reveal bloodmarks on the various porous and nonporous surfaces chosen in this study. Luminol. Amido Black and Crowle's Double Stain showed the highest sensitivity of all seven chemicals tested and revealed highly diluted (1:200) bloody fingerprints. Both luminol and Amido Black produced excellent results on both porous and nonporous surfaces, but Crowle's Double Stain failed to produce any results on porous substrates. Hungarian Red, DFO, leucomalachite green and ninhydrin showed lower sensitivities. Enhancement of bloodmarks using any of the chemicals selected, and short-term exposure to these same chemicals (i.e., less than 54 days), had no adverse effects on the PCR amplification of the nine STR systems surveyed (D3S 1358, HumvWA, HumFGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820) or of the gender determination marker Amelogenin. The intensity of the fluorescent signals was very similar and the allele size measurements remained constant and identical to those of untreated bloody fingerprints. No additional background fluorescence was noted. Continuous exposure (for 54 days) to two of the seven enhancement chemicals selected (i.e., Crowle's Double Stain and Hungarian Red) slightly reduced the amplification efficiency of the longer STR loci in profiles of fresh and 7 to 14-day-old bloodprints. This suggests that long-term exposure to these chemicals possibly affects the integrity of the DNA molecules. This study indicates that significant evidence can be obtained from fresh or aged bloody fingerprints applied to a variety of absorbent and nonabsorbent surfaces which are exposed to different enhancement chemicals for short or long periods of time. It also reaffirms that PCR STR DNA typing procedures are robust and provide excellent results when used in concert with fluorescence-based detection assays after fingerprint identification has taken place.     

Influence of the luminol chemiluminescence reaction on the confirmatory tests for the detection and characterization of bloodstains in forensic analysis

Preliminary tests for the detection of stains at crime scenes aim to focus the police work making them more efficient in the combat of criminality. The application of the luminol chemiluminescence reaction (3-aminoftalhidrazida) in presumptive tests for the detection of bloodstains is known for more than 40 years in forensic science. This reaction is based on the emission of light through the chemical reaction of luminol mixed with hydrogen peroxide and a hydroxide in the presence of a catalytic molecule (iron from the hemoglobin) (Laux [1]).This work evaluates the luminol interference and its effect on subsequent serological and DNA testing. Samples prepared with blood and different concentrations of luminol solution containing luminol, peroxide of hydrogen and sodium carbonate, were analyzed. Additionally, samples of serial dilutions of standard DNA mixed with luminol solution were also analyzed. Although presumptive tests with luminol do not establish the characterization and identification of stains at crime scenes, preliminary results indicated that it is suitable for the detection of invisible bloodstains for forensic analysis, with few detrimental effects on the serological tests and subsequent DNA recovery and typing.     

The effect of luminol on presumptive tests and DNA analysis using the polymerase chain reaction.

This study was designed to test the following factors involved with processing luminol treated bloodstained evidence: 1) The reactivity of other presumptive chemical color tests, phenolphthalin (PT) and tetramethylbenzidine (TMB), following the application of the light emitting luminol presumptive test. 2) The effect of different cleanings of various bloody substrates on the luminol test. 3) The effect of different cleanings of various bloody substrates on the ability to obtain DNA suitable for PCR testing. 4) The ability to extract DNA from luminol treated bloodstained substrates using three extraction techniques. 5) The effect of spraying washed and unwashed bloodstains on various substrates with luminol on the ability to correctly type the DNA using PCR. Our findings indicated that luminol did not adversely effect the PCR testing and did not interfere with the PT and TMB presumptive tests for blood. It was determined that the substrate and the method of cleaning were the major factors affecting DNA yield and the ability to type the bloodstains using PCR based technologies.     

The application of visible wavelength reflectance hyperspectral imaging for the detection and identification of blood stains

Current methods of detection and identification of blood stains rely largely on visual examination followed by presumptive tests such as Kastle–Meyer, Leuco-malachite green or luminol. Although these tests are useful, they can produce false positives and can also have a negative impact on subsequent DNA tests. A novel application of visible wavelength reflectance hyperspectral imaging has been used for the detection and positive identification of blood stains in a non contact and non destructive manner on a range of coloured substrates. The identification of blood staining was based on the unique visible absorption spectrum of haemoglobin between 400 and 500 nm. Images illustrating successful discrimination of blood stains from nine red substances are included. It has also been possible to distinguish between blood and approximately 40 other reddish stains. The technique was also successfully used to detect latent blood stains deposited on white filter paper at dilutions of up to 1 in 512 folds and on red tissue at dilutions of up to 1 in 32 folds. Finally, in a blind trial, the method successfully detected and identified a total of 9 blood stains on a red T-shirt.     

A Comparison of Chemical Enhancements for the Detection of Latent Blood,,

Luminol, Bluestar^®, and Hemascein^® were tested to compare detection sensitivities to latent blood. Untreated, EDTA‐treated human blood, and a catalytically similar blood substitute were diluted (neat to 1:1,000,000) and pipetted onto a variety of substrates. Luminol and Bluestar^® performed similarly on all surfaces and fabrics. Hemascein^® yielded poor results on wood surfaces, but performed well in the detection of latent blood on fabrics. Results from untreated, EDTA‐treated, and synthetic blood results indicate that EDTA‐treated blood is similar or slightly less sensitive than untreated blood at all dilutions and on all substrates, and the synthetic blood is less sensitive than real blood, but consistent in detection threshold and thus is useful as a training aid. Additionally, some foods and household chemicals that have previously been shown to cross‐react were tested with Bluestar^®, Hemascein^®, and luminol. Hemascein^® cross‐reacted with many substances, while both luminol reagents were more discriminating.     

Comparative performance of the Phadebas® Forensic Press Test at room temperature and 37 °C for the detection of saliva stains on fabric exhibits

The Phadebas® Forensic Press Test (PFPT) is an enzyme-based colorimetric test used to visualise and locate latent saliva stains on forensic exhibits. The test relies upon the presence of the enzyme α-amylase which is present in high levels in saliva. Even though the optimal in vitro temperature for α-amylase activity is 37 °C, the PFPT manufacturer’s protocol specifies that the PFPT should be carried out at room temperature (RT). In this study, we compared the performance of the PFPT at RT and 37 °C using combinations of four fabric types (cotton, polyester, acrylic and a cotton/polyester blend), three saliva dilutions (neat, 1:10 and 1:100) and stains aged for four time periods (1 day, 1 week, 1 month and 3 months). The intensity of the PFPT colour reactions at RT and 37 °C were not statistically different across all fabric types, saliva concentrations and stain ages, indicating that maximum sensitivity and performance of the PFPT can be achieved at RT.     

The effect of various stain carriers on the quality and quantity of DNA extracted from dried bloodstains

Bloodstains were made with 200 microliters blood on each of 11 different common substrates to examine the effect of the stain carrier on the amount and quality of DNA recoverable. High-molecular-weight DNA was extracted from all samples after 2 days. The yield of DNA from each sample varied considerably, not only between the different stain carriers but also within a given category. With a DNA yield of up to 10 micrograms, paper, glass, nylon, wood, smooth leather and wool gave the best results, followed by blue denim and wallpaper (up to 6 micrograms), cotton fabric and carpeting (up to 4 micrograms) and suede (up to 2 micrograms). For several stain carriers the DNA-containing solution was contaminated by chemical substances, which in the case of the blue denim, suede, and carpet samples inhibited the digestion of the DNA with restriction enzymes and prevented DNA typing. The different textures of the stain carriers tested and (as for varying yields on the same carrier) the differing degree of loss of DNA during extraction and the physiological variation in the number of leukocytes in human blood are discussed as possible reasons for the wide range of variation in the amounts of DNA it was possible to extract.     

A novel approach to obtaining reliable PCR results from luminol treated bloodstains

In recent years the forensic scientist has been afforded great advances in technology both in the detection of latent bloodstains and in acquiring reliable DNA typing results from very small pieces of physical evidence. Scientists are now able to detect minute quantities of latent bloodstains by utilizing the luminol reagent, oftentimes indicating that an attempt has been made to conceal any evidence of bloodshed. With the introduction of PCR based technology to the forensic arena, scientists are now routinely able to obtain DNA typing results from previously insufficient amounts of biological material, items as small as a single hair, saliva on a cigarette butt, or a bloodstain the size of a pin head. We present here a merging of these two advances coupled with a new collection medium for post luminol treated latent bloodstains. The forensic scientist is now able to routinely isolate and recover an adequate amount of DNA suitable for PCR typing at all of the Promega GenePrint PowerPlex 1.1 loci. In this study, several dilutions of latent bloodstains were prepared in an effort to simulate transferred bloodstains that are routinely encountered in a crime scene setting. The latent bloodstains were treated with luminol and subsequently collected using conventional cotton tipped swabs as well as a Puritan sponge tipped swab. PCR typing at the Promega GenePrint PowerPlex 1.1 loci was then attempted upon all dilutions of the latent bloodstains for both collection mediums. The results clearly indicate that it is now routinely possible to recover adequate amounts of DNA suitable for PCR typing upon post luminol treated bloodstains.     

A Comparison of Four Presumptive Tests for the Detection of Blood on Dark Materials

Detection of blood on dark materials is difficult for crime scene examiners so presumptive tests are used to assist. This study compared the ability of luminol, leuko crystal violet, tetramethylbenzidine, and Combur Test®E to detect whole, diluted blood (1:100) and a key‐shaped blood transfer stain (1:10), on dark cotton sheeting, tea towel, socks, synthetic carpet, and car mats. Powdered bleach was used to evaluate specificity of the blood detection tests. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall misclassification rate (OMR) assessed the quality of the blood tests. Luminol was the preferred test for diluted blood having the highest sensitivity (79%–96%), NPV (66%–93%), and the lowest OMR (3%–15%). Luminol was also found to be most efficient with a testing time on 25 items of 2 h 50 min compared with up to 8 h. Overall, luminol was the most effective method, also providing information on bloodstain patterns.     

Effect of "Fit" dishwashing detergent from former Eastern Germany (GDR) on luminol luminescence

The forensic luminol test is used to screen large areas for the presence of blood. The heme-induced reduction of hydrogen peroxide is coupled to the oxidation of luminol resulting in luminescence. However, photographic documentation of the relatively weak and short-lived luminescence is difficult and luminol is now often replaced by other chemicals. In this study, we investigated reports from the Rostock police department that the addition of "Fit", a dishwashing detergent from former Eastern Germany, could both intensify and prolong the luminescence of luminol on blood stains. Even though this effect was reported only for the original composition of Fit but not the currently sold version, we found that both the old and the new version of Fit increase the brightness of the luminescence while decreasing its duration. This may be due to detergents in the dishwashing liquid, which permeabilize the plasma membrane of the erythrocytes, exposing the Fe3+ inside the cell and speeding up the entire reaction. We did not find any evidence of special ingredients in the old version of Fit that would cause both the increased brightness and prolonged duration of luminescence as reported by the Rostock PD.     

Determination of postmortem interval from old skeletal remains by image analysis of luminol test results.

The luminol test is routinely used in forensic serology to locate blood traces and identify blood stains not visible to the naked eye; its sensitivity is reported as ranging from 1:100,000 to 1:5,000,000. To evaluate the possibility of correlating the postmortem interval with blood remnants in bone tissue, the luminol test was performed on 80 femurs with a known time of death, grouped in five classes. Powdered bone (30 mg) was recovered from compact tissue of the mid-shaft of each femur and was treated with 0.1 mL of Luminol solution (Sirchie Finger Print Laboratories, Inc.). The reactions were observed in a dark room and filmed by a TV camera equipped with a recording tape. An intense chemiluminescence was observed after a few seconds in all 20 femurs with a PMI ranging from 1 month to 3 years. On the 20 femurs with a PMI ranging from 10-15 years, a clear chemiluminescence was visible with the naked eye in 80% of the sample. Among the 20 femurs with a PMI ranging from 25 to 35 years, a weaker chemiluminescence appeared in 7 femurs (33% of the sample). In the 10 femurs with a PMI ranging from 50 to 60 years, a faint reaction was observed only in a single femur. In none of the ten femurs with a PMI over 80 years was chemiluminescence observed. The image of each reaction was computerized and analyzed for gray scale. The results of image analysis show a possible quantitative relationship between the PMI and luminol chemiluminescence in powdered bone.     

Postmortem interval of skeletal remains through the detection of intraosseal hemin traces. A comparison of UV-fluorescence, luminol, Hexagon-OBTI®, and Combur® tests

With the goal of obtaining additional practically applicable methods for estimating the PMI of skeletal remains, 39 samples of human and 5 samples of domestic animal long bones with known PMI (PMI=1 to approximately 2000 years) were tested with two established methods (UV-fluorescence of a freshly sawn cross-section and the luminol test) and two screening tests (Hexagon-OBTI? test and Combur? test) that were being tried out in this context for the first time. The hypothesis underlying this experiment was the supposition that the PMI-related chemiluminescence of the luminol reaction for bone is based on the presence of persisting hemin from hemoglobin molecules in bone. Our results showed that lack of luminescence and reduced UV-fluorescence were more meaningful results for estimating PMI and excluding forensic relevance than a positive luminol reaction or strong UV-fluorescence, as both of the latter findings revealed the limitations of these methods in this particular context. Particularly for cases showing a positive luminol reaction, the use of additional absolute dating methods may be indicated. Against our expectations, both the Combur? test strips and the Hexagon-OBTI? test, which were both devised to demonstrate blood, delivered negative results for all samples. They are thus not suitable for estimating the PMI of skeletal remains. Future research will be necessary to elucidate whether the negative results obtained for these tests may be due to the poor solubility of potentially present hemoglobin or hemoglobin breakdown products in the Tris buffer used in this experiment.     

The killer outfit and timing: Impact of the fabric and time in body fluid identification and DNA profiling

The present work aimed to study the detection, through lateral flow immunochromatographic (LFI) tests, of saliva samples over time in three different types of fabrics, as well as, the possibility of DNA isolation and characterization from the sample tubes and the cassettes. Fifty microliters of saliva (three samples/time) were deposited in denim, cotton, and polyester. Saliva was identified by SERATEC® Amylase Test and the Crime Scene version SALIVA CS, being able to detect it up to six months of deposition, although with different band intensities. Polyester showed stronger bands than cotton, probably due to its synthetic nature, and denim, as an inked fabric, showed less band intensities. Statistical analyses confirmed significant differences among fabrics, but not over time in the same type of fabric. Total DNA from the sample tubes was successfully recovered, in contrast, from the cassettes, only polyester retrieved amplifiable DNA. These findings indicated that it is possible to recover and identify saliva up to six months after deposition, also obtaining DNA. Future research will be able to expand these results, analyzing the stability of other body fluids, and the sensitivity of lateral flow immunochromatographic tests to detect them.     

Validation studies of an immunochromatographic 1-step test for the forensic identification of human blood.

An immunochromatographic 1-step test for the detection of fecal occult blood was evaluated for applicability for the forensic identification of human blood in stained material. The following experiments were conducted: 1) determination of the sensitivity and specificity of the assay; 2) evaluation of different extraction media for bloodstains (sterile water, Tris buffer pH 7.5 provided in the test kit, 5% ammonia); 3) analysis of biological samples subjected to a variety of environmental insults; and 4) evaluation of casework samples. This immunochromatographic 1-step occult blood test is specific for human (primate) hemoglobin and is at least an order of magnitude more sensitive than previous methods for detecting human hemoglobin in bloodstains. The antigen is insensitive to a variety of environmental insults, except for exposure to certain detergents and household bleaches and prolonged exposure to certain preparations of luminol. The entire assay can be conducted in field testing conditions within minutes. When in the laboratory the supernatant from a DNA extraction is used for the assay, there is essentially no consumption of DNA for determining the presence of human hemoglobin in a forensic sample. The data demonstrate that this test is robust and suitable for forensic analyses.     

Developmental Validation of the PrepFiler™ Forensic DNA Extraction Kit for Extraction of Genomic DNA from Biological Samples*

Abstract: The PrepFiler? Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 μL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA^® paper), and touch evidence‐type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol–chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.