Comparison of methods for visualizing blood on dark surfaces

Difficulties can arise when screening dark casework items for blood, a poor contrast between blood and the background can mean stains are not always evident. Typical indirect searching methods can be time consuming and may result in potentially important bloodstains being missed. Luminol, fluorescein, hydrogen peroxide, ultraviolet light and infrared photography were tested in an effort to find a rapid and efficient blood search tool for direct application to dark surfaces. Methods were compared in their sensitivity, specificity, ability to work on various surface types and their effect on DNA extraction and typing. Along with experimental results, the ease of use, costs and the health and safety considerations were also compared. Hydrogen peroxide was determined to be the most effective method. However, where blood was likely to be dilute, luminol was proposed due its greater sensitivity.     

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.     

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.     

Evaluation of six presumptive tests for blood, their specificity, sensitivity, and effect on high molecular-weight DNA

Luminol, leuchomalachite green, phenolphthalein, Hemastix, Hemident, and Bluestar are all used as presumptive tests for blood. In this study, the tests were subjected to dilute blood (from 1:10,000 to 1:10,000,000), many common household substance, and chemicals. Samples were tested for DNA to determine whether the presumptive tests damaged or destroyed DNA. The DNA loci tested were D2S1338 and D19S433. Leuchomalachite green had a sensitivity of 1:10,000, while the remaining tests were able to detect blood to a dilution of 1:100,000. Substances tested include saliva, semen, potato, tomato, tomato sauce, tomato sauce with meat, red onion, red kidney bean, horseradish, 0.1 M ascorbic acid, 5% bleach, 10% cupric sulfate, 10% ferric sulfate, and 10% nickel chloride. Of all the substances tested, not one of the household items reacted with every test; however, the chemicals did. DNA was recovered and amplified from luminol, phenolphthalein, Hemastix, and Bluestar, but not from leuchomalachite green or Hemident.     

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.     

Evaluation of Bluestar® Forensic Magnum and Other Traditional Blood Detection Methods on Bloodstained Wood Subjected to a Variety of Burn Conditions,

Accurate blood detection is a primary concern for forensic scientists, especially in highly compromised situations. In this study, blood was added to wood blocks and subjected to a variety of fire treatments: the absence or presence of accelerant, burn time (1, 3, or 5 min), and extinguishment method (smothering or dousing with water). Burned blocks were given a qualitative burn score, followed by removal of half of the char from each block and subsequent testing of each half for blood using luminol (13% positive; n = 96), Bluestar^® Forensic Magnum (5.2% positive; n = 96), and combined phenolphthalein tetramethylbenzidine test (0% positive; n = 192). Luminol and Bluestar® Forensic Magnum performed similarly, both outperforming PTMB. Additionally, positive results were more likely from samples that were smothered, had a low burn score, and had more concentrated blood solutions (neat or 1:2). Overall, it is extremely unlikely that blood would be detected on combustible substrates exposed to direct fire.     

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.     

Photoluminescence of blood by acidic hydrogen peroxide—A preliminary test

In this study, the authors found that treating blood with 1 M HCl and 2% (w/v) 5-sulfosalicylic acid (SSA) in 1% (v/v) hydrogen peroxide mixture can produce photoluminescence of blood. SSA was added as a blood fixer. The photoluminescence was induced by irradiation of a forensic light source at 505 nm, which was detected using a 550 nm barrier filter. In this experiment, various level of acid and hydrogen peroxide were tested to find the optimal formulation of reagents, spot tests were conducted with diluted blood to test the sensitivity of this reagent, and impressions in blood left on porous/nonporous surfaces were enhanced. The sensitivity of this solution was slightly lower than Bluestar and was similar to leucocrystal violet or leucomalachite green on both porous/non-porous surfaces. The photoluminescence of blood treated with this reagent has been observed over 2 months. Using this reagent, it was possible to observe fingermarks or footwear impressions in blood on a black porous/non-porous surface. Through this, it was found that using this reagent could enhance bloodstains regardless of the porosity or color of the surface.     

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.     

Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) detection limits for blood on fabric: Orientation and coating uniformity effects

Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) was used to analyze four types of forensically relevant fabrics coated with varying dilutions of blood. The blood was applied in two manners, dip coating with a smooth and uniform layer and drip coating with droplets from pipettes. Spectra of neat and dip coated fabrics were acquired using controlled orientations, and these were compared to spectra collected on samples with random orientations. The improved reproducibility seen in visual inspection of the spectra is confirmed by principal component and linear discriminant projections of the spectra, as well as by statistical hypothesis testing. Principal component regression (PCR), using the regions of the IR spectra associated with the amide A/B, I, II, and III vibrational bands (3500–2800, 1650, 1540, and 1350 cm⁻¹), was employed on the more uniform dip coated spectra to estimate limits of detection for blood on two of the four fabrics – acrylic and nylon. These results demonstrate that detection limits for blood on fabrics can be decreased significantly by controlling for the orientation and face of the fabric samples while collecting spectra. Limits of detection for acrylic and nylon were found to be 196 × and 227 × diluted blood, respectively.     

idPAD: Paper Analytical Device for Presumptive Identification of Illicit Drugs

As drug overdose deaths across the United States continue to rise, there is increasing interest in field testing of illicit substances. This work discusses a paper-based analytical device (idPAD) that can run a library of 12 colorimetric tests at the same time, each detecting different chemical functional groups and materials found in illicit drugs, distractor substances, and cutting agents. The idPAD requires no electricity, costs less than $2 USD, and requires minimal training to operate. The results of the 12 tests form a color barcode which is “read” by comparison to standard images. The accuracy of the idPAD was assessed using samples of heroin, cocaine HCl, crack, and methamphetamine at concentrations of 25%–100% in a lactose matrix, as well as pure lactose. Based on 840 “reads” by three different users, the idPAD showed 95% sensitivity and 100% specificity for detecting these drugs; the most common error was mistaking cocaine HCl for crack or crack for cocaine HCl. In a second step, samples of heroin, cocaine, and methamphetamine (n = 30) and distractor substances (pharmaceuticals, cutting agents, and other illicit drugs, n = 64) were tested by two readers, yielding a sensitivity of 100% and specificity of 97%. Targeted substances were detected reliably at 55–180 μg/lane, and the idPAD was found to be stable for at least 3 months when stored at room temperature. The library approach used in the idPAD may provide the accuracy and robustness necessary for a presumptive field drug test.     

Variability in the Blood/Breath Alcohol Ratio and Implications for Evidentiary Purposes

The breath analyzer is an indispensable tool for identifying alcohol levels among drivers. While numerous studies have shown high correlations between blood and breath alcohol concentrations, most are limited by the study design. This study seeks to assess this relationship by minimizing potential measurement bias, document time from alcohol consumption to testing, and adjusting for potential confounders. A blinded study was performed using conditions closely resembling those in the field. The Draeger 7110 MKIII IL breath analyzer was used to assess breath alcohol concentrations (BrAC). Participants were 61 healthy volunteers aged 21–37 years with body mass index ≤30 and no history of alcoholism. A total of 242 valid blood/breath tests were performed in four test sets. The study results showed a high correlation coefficient between BrAC and blood alcohol concentration (BAC) levels (r = 0.983) with high sensitivity (97%) and specificity (93%). This strong association between the breath analyzer and BAC persisted even after adjustment for various stages of alcohol absorption. These results illustrate the high diagnostic sensitivity of the breath analyzer in field‐tested conditions.     

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.     

A comprehensive study into false positive rates for ‘other’ biological samples using common presumptive testing methods

The identification of biological fluids or materials in forensic samples is a key requirement in forensic science that relies on chemical and biological based tests, most of which exhibit false positivity. When reporting results from such tests, Forensic Scientists use words such as probable, possible, and likely, without always being able to provide robust support for these conclusions. In collating information about false positive rates for a number of these tests, we found limited research into the cross reactions observed from ‘other’ biological samples in commonly encountered case sample stains. By ‘other’ we mean biological fluids or materials that are not the primary target of the presumptive test being used. Here we carry out a specificity study to fill gaps in the literature for a number of the presumptive chemical, biological and immunochromatographic tests used to presumptively screen for blood, semen and saliva. The tests selected for this study are the widely used tests: Luminol, TMB/Combur³ Test® E, Kastle-Meyer (KM), RSID™ - Blood, ABAcard® HemaTrace®, Acid Phosphatase (AP), ABAcard® p30, RSID™ - Semen, Phadebas® ‘Tube’ Test, Phadebas® ‘Press’ Test, and RSID™ - Saliva tests. Specificity for each of these was tested in known samples, from volunteers, of blood, semen, saliva, urine, sweat, vaginal material, faeces and breast milk, and then false positive rates were determined.     

Detection of acute fentanyl exposure in fresh and decomposed skeletal tissues part II: The effect of dose–death interval

The effects of dose–death interval on the detection of acute fentanyl exposure in fresh and decomposed skeletal tissues (marrow and bone), by automated enzyme-linked immunosorbent assay (ELISA) are described. Rats (n = 14) were administered fentanyl acutely at a dose of 0 (n = 2) or 60 μg/kg (n = 12) by intraperitoneal injection, and euthanized within 20, 45, 135, or 225 min. Femora and tibiae were extracted from the fresh corpses and marrow was isolated from the femoral and tibial medullary cavities. The remains were then allowed to decompose outdoors to the point of complete skeletonization, and vertebrae, pelvi and miscellaneous (humeri and scapulae) were recovered for analysis. In all cases, bones were cleaned in alkaline solution and then ground into a fine powder. Marrow was homogenized in alkaline solution. Fentanyl was extracted from ground bone by methanolic extraction. Extracts were adjusted to pH 6 and analyzed by ELISA. Perimortem heart blood was also collected and diluted in phosphate buffer prior to screening by ELISA. The effect of tissue type on ELISA response was examined through determination of binary classification test sensitivity and the relative decrease in absorbance (%DA, drug-positive tissues vs. drug-free controls) in each tissue type. Overall, the %DA varied significantly between extracts from different skeletal tissues at a given dose–death interval, according to the general order of marrow > decomposed bone > fresh bone. Binary classification test sensitivity values for fentanyl in marrow, fresh epiphyseal (femoral and tibial) bone, fresh diaphyseal (femoral and tibial) bone, decomposed vertebrae, decomposed pelvic bone, and decomposed miscellaneous bone were 67–100%, 0–33%, 0–33%, 0–67%, 0–67% and 0–33%, respectively, over all dose–death intervals. Although group mean %DA values showed a strong negative correlation with dose–death interval in marrow, fresh epiphyseal bone, decomposed vertebrae, pelvic and miscellaneous bone (r = ?0.989, ?0.930, ?0.955, ?0.903, and ?0.974, respectively), the high variability in both fresh and decomposed bone precluded differentiation of the dose–death intervals based on %DA value alone. Overall, the results suggested that the type of skeletal tissue sampled may not be as important as the amount of residual marrow remaining in skeletonized remains.     

A novel method for cyanide quantification in human whole blood using ion chromatography with amperometric detection and its application to cyanide intoxication cases

Cyanide is a highly toxic agent that has been frequently used for suicide in South Korea. It is also used in various industrial fields, such as metal plating, in which many accidental cyanide intoxications have occurred. To overcome the disadvantages of conventional cyanide analysis methods, a simple and fast method for the analysis of cyanide in whole blood using ion chromatography (IC) with amperometric detection was developed in this study. Whole blood samples were deproteinized, diluted, and analyzed using an IC–amperometric detection system. The limits of detection and quantitation were 0.1 and 0.2 mg/L, respectively. The method showed good linearity in the range of 0.2 to 50 mg/L with R² > 0.99. The intra- and inter-assay precision and accuracy values were <10%. The established method was successfully applied to analyze whole blood samples from three cyanide intoxication cases.     

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.     

Distal Humerus Morphological Variation and Sex Estimation in Modern Thai Individuals,

Due to differential preservation, it is necessary to develop sex estimation methods on varied anatomical regions, including the distal humerus. Sexually dimorphic differences of the medial epicondyle angle, olecranon fossa shape, trochlear extension, and trochlear constriction have been documented in several non-Asian groups. This study examines distal humerus morphological variation in 616 modern Thai individuals (f = 198; m = 418) 18–96 years old and tests the methods on a hold-out sample of 152 individuals (f = 91; m = 61). The results indicate that population-specific binary probit regression performs the best (74.1–100%), followed by composite scoring (77.0–90.1%), binary logistic regression (63.3–92.2%), and univariate sectioning points (37.7–90.1%). Age minimally effects the expression of the medial epicondyle angle in males, and trait scoring is susceptible to high intra- and interobserver error. While demonstrating relatively high sex biases, distal humerus morphology can be used to estimate the sex of Thai individuals when more sexually dimorphic regions are absent.     

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.     

Illuminating the health and safety of luminol

Luminol is a reagent that is used to enhance areas of non-visible bloodstaining and it is one of the most sensitive of such reagents available to the forensic scientist. However, its use, particularly within the UK and some other European countries, has been limited, predominantly due to concerns about the health and safety of the reagent. This paper reviews the literature currently available regarding the health and safety of luminol, and in the authors' view demonstrates that there are no significant health and safety concerns with the preparation of luminol solution and its application at the crime scene or in the laboratory, providing suitable precautions are taken.