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.     

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.     

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.     

Phenolphthalein False‐Positive Reactions from Legume Root Nodules

Presumptive tests for blood play a critical role in the examination of physical evidence and in the determination of subsequent analysis. The catalytic power of hemoglobin allows colorimetric reactions employing phenolphthalein (Kastle‐Meyer test) to indicate “whether” blood is present. Consequently, DNA profiles extracted from phenolphthalein‐positive stains are presumed to be from blood on the evidentiary item and can lead to the identification of “whose” blood is present. Crushed nodules from a variety of legumes yielded phenolphthalein false‐positive reactions that were indistinguishable from true bloodstains both in color quality and in developmental time frame. Clothing and other materials stained by nodules also yielded phenolphthalein false‐positive reactivity for several years after nodule exposure. Nodules from leguminous plants contain a protein (leghemoglobin) which is structurally and functionally similar to hemoglobin. Testing of purified leghemoglobin confirmed this protein as a source of phenolphthalein reactivity. A scenario is presented showing how the presence of leghemoglobin from nodule staining can mislead investigators.     

The possibility for determining haptoglobin phenotypes in blood stains after treatment with a luminol solution

The paper gives the results of tests for influence of luminol solution of different composition on detectability of haptoglobin fractions in the bloodstains of different ages. It was stated that alkaline luminol solutions reduce intensity of fractions and may hamper Hp phenotype determination especially in old stains.     

Black and green tea - luminol false-negative bloodstains detection

The antioxidant properties of black and green teas are well known. It is also possible to determine their antioxidant capacity by using a chemiluminscent method. This method is based on the measurement of the delay in the emission of light from the luminol reaction in the presence of the antioxidant. Bloodstains which are invisible to the naked eye can also be detected by luminol. Three common methods (detection using the Grodsky or Weber formulations and by Bluestar® Forensic latent bloodstain reagent) are based on the luminol chemiluminescence reaction. The bloodstains can be masked by drinks and/or foods containing antioxidants. The aim of this work was to compare the ability of black and green teas containing antioxidants to cause false negative results during chemiluminescent bloodstain detection.     

The Reliability of Pattern Classification in Bloodstain Pattern Analysis—PART 2: Bloodstain Patterns on Fabric Surfaces,

This study was designed to produce the first baseline measure of the reliability of bloodstain pattern classifications on fabric surfaces. Experienced bloodstain pattern analysts classified bloodstain patterns on pairs of trousers that represented three fabric substrates. Patterns also varied in type (impact, cast‐off, expiration, satellite stains from dripped blood, and transfer) and extent. In addition, case summaries that accompanied each pattern contained contextual cues that either supported the correct answer (i.e., positive bias), were misleading toward an incorrect answer (i.e., negative bias), or contained no directional information (i.e., neutral). Overall, 23% percent of the resulting classifications were erroneous. The majority (51%) of errors resulted from analysts misclassifying satellite stains from dripped blood. Relative to the neutral information, the positive‐bias information increased correct classifications and decreased erroneous classifications, and the negative‐bias information decreased correct classifications and increased erroneous classifications. The implications of these findings for BPA are discussed.     

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.     

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.     

PCR扩增ZFY/ZFX基因鉴定人类干血痕性别

应用 PCR 技术同时扩增人 ZFY 和 ZFX 基因特异的 DNA 序列,在男性血痕中可检测到两种扩增产物,即340bp 长的 ZFY 基因及488bp 长的 ZFX 基因特异 DNA 片段;在女性血痕中仅可检测到488bp 长的 ZFX 基因特异 DNA 片段,据此判定干血痕性别。干血痕的最小检出需要量为0.125μl 血液量的血痕。室温保存10年的血痕可以准确判定性别。ZFY 基因位于 Y 染色体短臂。本方法同时检测两条性染色体,可以避免由于扩增失败或 Y 染色体长臂变异出现的假阴性或假阳性。扩增产物经琼脂糖凝胶电泳即可区分。     

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.     

Application of mRNA Expression Analysis to Human Blood Identification in Degenerated Samples that were False‐negative by Immunochromatography,,

Forensic laboratories are often faced with cases in which methamphetamine hydrochloride‐mixed blood is unable to be identified as human blood by immunochromatography against human hemoglobin A0. The application of mRNA expression analysis to samples that showed a false‐negative with immunochromatography was investigated as an alternative approach that did not depend on the antigen–antibody reaction. Real‐time PCR was used to examine the expression levels of blood markers such as glycophorin A, spectrin beta, and hemoglobin beta. Hemoglobin beta was the only marker that was specifically detected in blood, while glycophorin A was useful for determining human specificity. Hemoglobin beta showed good detection sensitivity and was detectable in 37‐year‐old blood stains. Hemoglobin beta was exclusively detectable in methamphetamine hydrochloride‐mixed blood stains. Detergents and disinfectants did not significantly influence mRNA markers. The proposed mRNA expression analysis was suitable for human blood identification as an alternative method to immunochromatography.     

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.     

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.     

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.     

Forensic Analysis of Stains on Fabric Using Direct Analysis in Real‐time Ionization with High‐Resolution Accurate Mass‐Mass Spectrometry

A rapid technique using direct analysis in real ‐ time (DART) ambient ionization coupled to a high ‐ resolution accurate mass‐mass spectrometer (HRAM‐MS) was employed to analyze stains on an individual's pants suspected to have been involved in a violent crime. The victim was consuming chocolate ice cream at the time of the attack, and investigators recovered the suspect's pants exhibiting splatter stains. Liquid chromatography with mass spectral detection (LC‐MS) and stereoscopic light microscopy (SLM) were also utilized in this analysis. It was determined that the stains on the pants contained theobromine and caffeine, known components of chocolate. A shard from the ceramic bowl that contained the victim's ice cream and a control chocolate ice cream sample were also found to contain caffeine and theobromine. The use of DART‐HRAM‐MS was useful in this case due to its rapid analysis capability and because of the limited amount of sample present as a stain.     

Detection of semen and blood stains using polilight as a light source.

Photoluminescence spectra of dry untreated semen have been measured and a suggested method for rapid detection of untreated semen stains is derived from these measurements. The method is presented in the form of a flow chart to cover most crime scene situations. The absorption spectrum of dry untreated blood has also been measured and a suggested method for enhancement and photography of blood stains is derived from this measurement. The method is presented in the form of a flow chart. Both methods are based on the use of a high intensity light source such as the Polilight.     

A Comparison of DNA Extraction Using AutoMate Express™ and EZ1 Advanced XL from Liquid Blood,Bloodstains, and Semen Stains

In this study, DNA was extracted using an AutoMate Express? and an EZ1 Advanced XL from liquid blood, fresh and aged bloodstains, and fresh and aged semen stains. Extracted DNA was quantified by real‐time PCR using the D17Z1 locus. Short tandem repeat typing was performed using an AmpF?STR^® Identifiler kit. The yields of DNA obtained by the AutoMate Express? were higher from fresh bloodstains and fresh semen stains, almost the same from aged bloodstains and aged semen stains, but slightly lower from liquid blood compared with those obtained by the EZ1 Advanced XL. The addition of dithiothreitol or the use of PrepFiler? lysis buffer improved the EZ1 Advanced XL results from fresh bloodstains, but not for liquid blood and aged bloodstains. Our results demonstrated that the PrepFiler? lysis buffer is the main contributor to the higher DNA yields of the AutoM ate Express? for fresh bloodstains.     

The Effect of Household Oxidizing Cleaners on Chemiluminescence of Blood Using Bluestar®

This study tests the effect of three common oxidizing cleaners on the ability of the Bluestar Forensic® presumptive test for blood to identify the presence of blood on ceramic tile after cleaning. The cleaners tested were Lysol®, OxiClean®, and Arm & Hammer®. This study also tested which cleaner was the most effective at removing blood, measured by the intensity of chemiluminescence, which was quantified using RGB values in ImageJ. A “hasty” 1‐min cleaning of a blood droplet was simulated using the three cleaners. The chemiluminescence of the Bluestar® reactions after cleaning the blood‐treated region was compared to an untreated region of the same tile for each cleaner, as well as to the treated regions of tiles between the three cleaners. Results indicate that none of the three cleaners removed all of the blood (all p < 0.001) and that Lysol® removed more blood compared to the OxiClean® and Arm & Hammer®.