Barbara Daniel

Comparison of IgE and IgG antibody-dependent cytotoxicity in vitro and in a SCID mouse xenograft model of ovarian carcinoma.

Allergic reactions are mediated by IgE antibodies bound to high‐affinity receptors on mast cells in peripheral tissues and are characterized by their immediacy and hypersensitivity. These properties could also be advantageous in immunotherapy against cancer growth in peripheral tissues. We have constructed chimeric IgE and IgG1 antibodies with murine V regions and human C regions corresponding to the MOv18 monoclonal antibody against the human ovarian tumor‐associated antigen, folate binding protein. The antibodies exhibited the expected binding affinities for antigen and Fc receptors, and effector activities with human basophils and platelets in vitro. The protective activities of MOv18‐IgE and MOv18‐IgG1 were compared in a SCID mouse xenograft model of ovarian carcinoma. The beneficial effects of MOv18‐IgE were greater and of longer duration than those of MOv18‐IgG1. Our results suggest that the allergic reaction could be harnessed for the suppression of ovarian tumors.

Recovery of DNA and fingerprints from touched documents

This study investigated the various factors affecting DNA profiling from DNA recovered from fingerprints deposited on paper before and after fingerprint enhancement treatments. The DNeasy plant mini kit (QIAGEN) was found to improve DNA recovery from paper by over 150% compared with the QIAamp mini kit. A significant decrease in the amount of DNA recovered was observed following treatment with DFO and/or Ninhydrin. This decrease in yield did not have a comparably significant effect on the quality of the SGM Plus profiles. Furthermore, this study found that whilst certain paper types, such as newspaper, magazine and filter paper allowed for the good recovery of DNA, common office paper and white card, strongly interfered with the recovery of DNA resulting in poor quality profiles.

Comparison of the effects of sterilisation techniques on subsequent DNA profiling.

It is important that contamination from extraneous DNA should be minimised on items used at crime scenes and when dealing with exhibits within the laboratory. Four sterilisation techniques (UV, gamma and beta radiation and ethylene oxide treatment) were examined for their potential to degrade contaminating DNA to such an extent that subsequent DNA profiling was impossible. This work indicated that the most successful technique to reduce DNA contamination was ethylene oxide treatment. Of the radiation techniques tested in this study, gamma was the most successful at eradicating DNA and UV radiation was the least. None of the contaminated samples treated with ethylene oxide and subsequently subjected to DNA analysis met the DNA profile criteria necessary for acceptance on the UK National DNA Database. Contaminated cotton swabs and micro-centrifuge tubes treated with ethylene oxide showed a marked decrease in amplifiable DNA post-treatment. Ethylene oxide treatment to sterile swabs and tubes did not significantly affect subsequent DNA analysis.

Comparison of Presumptive Blood Test Kits Including Hexagon OBTI

Abstract:  Four presumptive blood tests, Hexagon OBTI, Hemastix®, Leucomalachite green (LMG), and Kastle‐Meyer (KM) were compared for their sensitivity in the identification of dried bloodstains. Stains of varying blood dilutions were subjected to each presumptive test and the results compared. The Hexagon OBTI buffer volume was also reduced to ascertain whether this increased the sensitivity of the kit. The study found that Hemastix® was the most sensitive test for trace blood detection. Only with the reduced buffer volume was the Hexagon OBTI kit as sensitive as the LMG and KM tests. However, the Hexagon OBTI kit has the advantage of being a primate specific blood detection kit. This study also investigated whether the OBTI buffer within the kit could be utilized for DNA profiling after presumptive testing. The results show that DNA profiles can be obtained from the Hexagon OBTI kit buffer directly.

Forensic DNA methylation profiling - Potential opportunities and challenges

Investigating the DNA sequence is the most powerful tool that can be employed in forensic genetics for the identification of an individual, or to determine specific ethnic and phenotypic characteristics. However, there are also other heritable changes in gene function or cellular phenotype which are caused by mechanisms other than differences in the DNA sequence itself. Over the last decade it has become evident that epigenetic markers can be of substantial forensic significance. The determination of possible alterations in DNA methylation patterns could aid various forensic investigations, such as differentiating monozygotic twins, identifying the tissue source or determining the age of tissue donors. This review aims to give a brief overview of the possible advantages of forensic DNA methylation profiling and sheds light on the limitations of this approach.

Wet powder suspensions as an additional technique for the enhancement of bloodied marks

The enhancement of marks in blood on dark surfaces poses significant challenges to the forensic scientist. Current methods of enhancement include the sequential use of acid dyes (acid yellow, acid violet and acid black). Acid yellow is used to greatest effect on lighter deposits of blood on a non-porous background, and is visualised using a light source which causes it to fluoresce [1]. However, further enhancement with acid violet and acid black produces a dark product which may fail to improve the contrast of the mark against a dark background. The use of wet powder suspensions (WPSs) has been proposed as a complementary procedure for use in fingermark enhancement, beyond its typical use in the enhancement of marks on adhesive surfaces. In this investigation, the use of WPS was tested in conjunction with conventional acid dye treatments on marks in blood deposited on a selection of substrates. The results demonstrated that white WPS alone or together with acid dyes results in an overall enhancement of mark quality (p<0.005) on marks deposited on smooth non-porous surfaces. The technique was shown to not interfere with subsequent presumptive tests on blood. However WPS treatments were shown to reduce the amount of DNA recoverable from the marks, resulting on an average decrease of 91% compared to untreated controls. The decline in DNA yields was shown to result in a decrease in the quality of the DNA profiles obtained. The enhancement properties of WPS were evaluated by electron microscopy. It was shown that the titanium dioxide particles in the WPS primarily interact with the non-bloodied part of the mark, thus producing a contrasting effect with the background and acid dyes.

Evaluation of semen presumptive tests for use at crime scenes

The SERATEC® PSA Semiquant and RSID®-Semen tests are immunoassay kits that identify semen by detecting prostate-specific antigen (PSA) and semenogelin (Sg), respectively. Both kits were tested with semen, urine, blood, saliva, vaginal secretions and breast milk in order to determine their sensitivity and specificity. These results demonstrate that the SERATEC PSA kit is more sensitive than the RSID-Semen kit with a limit of detection of 200 ng/mL as opposed to 8.0 × 103ng/mL. The RSID-Semen kit gave no false-positives or -negatives compared with 2.9% false-negatives with the SERATEC PSA kit. Results from postcoital samples show the RSID-Semen kit to be more effective, indicating that this kit is more suitable for semen identification in the Haven Suites. As a more robust and cost-effective kit, the SERATEC PSA test is recommended for use at crime scenes. The ability to obtain DNA profiles from the buffer of both kits demonstrates the potential benefit of these kits in a rape investigation. The use of these kits at crime scenes would provide an invaluable contribution by prioritizing samples for subsequent analysis, thereby allowing greater efficiency with investigation times.

A novel fluorescence-based method in forensic science for the detection of blood in situ

Full DNA profiles can be generated from just a few cells; however these profiles can be contaminated from other cell types present at the crime scene. We report here on the development of an immunofluorescent technique to spatially locate human-specific blood in situ and also on the ability of this technique to detect individual leukocytes and the DNA contained within them. Four monoclonal mouse anti-human antibodies were evaluated; anti-glycophorin A to detect erythrocytes and anti-CD45, anti-myeloperoxidase (MPO) and anti-histone H1 to detect the nucleated leukocytes. Each antibody was labeled with either Alexa Fluor 488 or 568 for direct application to blood smears which allowed the simultaneous detection of erythrocytes and leukocytes. Furthermore, because histones are DNA binding proteins, the application of anti-histone H1 allowed the detection of DNA within a blood smear. Importantly it was found that full DNA profiles could be achieved after using this method with similar peak area ratios compared to untreated cells. The fluorescent antibodies were found to be human-specific with the exception of anti-histone H1 due to its conserved sequence. However, used in combination with anti-CD45 or anti-MPO the location of DNA from human-specific leukocytes could be detected. The technique was also tested on older blood stains and was still found to be sensitive and cell-specific after 4 months. Following the optimization of the methodology, the fluorescent antibodies were applied to short lengths of black cotton fibres covered with human blood spots. Although the background fluorescence from the cotton was found to be high, erythrocytes and even individual leukocytes could easily be detected, indicating that this technique could be used to detect extremely minute amounts of blood. Used in combination with laser capture microdissection (LCM), this method could be used to pick off individual leukocytes for LCN DNA techniques.

Enabling fluorescent biosensors for the forensic identification of body fluids

The search for body fluids often forms a crucial element of many forensic investigations. Confirming fluid presence at a scene can not only support or refute the circumstantial claims of a victim, suspect or witness, but may additionally provide a valuable source of DNA for further identification purposes. However, current biological fluid testing techniques are impaired by a number of well-characterised limitations; they often give false positives, cannot be used simultaneously, are sample destructive and lack the ability to visually locate fluid depositions. These disadvantages can negatively affect the outcome of a case through missed or misinterpreted evidence. Biosensors are devices able to transduce a biological recognition event into a measurable signal, resulting in real-time analyte detection. The use of innovative optical sensing technology may enable the highly specific and non-destructive detection of biological fluid depositions through interaction with several fluid-endogenous biomarkers. Despite considerable impact in a variety of analytical disciplines, biosensor application within forensic analyses may be considered extremely limited. This article aims to explore a number of prospective biosensing mechanisms and to outline the challenges associated with their adaptation towards detection of fluid-specific analytes.

Development of a biosensor for human blood: new routes to body fluid identification.

The identification of human blood at a crime scene can provide crucial information to an investigation whilst also providing a source of nuclear material which can be targeted for DNA profiling. Here, we report on the development of an immunofluorescent biosensor for the identification of human blood which has the potential to overcome the drawbacks of the current body fluid identification techniques. An antibody (Ab) raised against human erythrocytes was conjugated to fluorescent semiconductor quantum dots (QDs) by sulfhydryl chemistry. The conjugation was verified by agarose gel electrophoresis and immunohistochemistry. Incubation of liquid blood samples with the conjugated nanocrystals was shown to quench the fluorescence emission spectra in a concentration-dependent manner. A different effect was observed with unconjugated QDs incubated in blood. Full profiles were obtained from blood samples previously treated with the Ab–QDs, demonstrating that the method does not interfere with DNA profiling. To our knowledge, this is the first example of a hybrid Ab–QD sensor that has the potential to be employed for the identification of human blood. The results of this study are expected to open up a new research direction in the field of body fluid detection.