Julianne Henry

Comparison of myeloma cell contamination of bone marrow and peripheral blood stem cell harvests

It could be speculated for patients with myeloma and other lymphoproliferative disorders that peripheral blood stem cells may be preferable to bone marrow for autologous transplantation because they may be less contaminated by neoplastic cells. To test this possibility, the immunoglobulin heavy chain gene rearrangement and limiting dilution polymerase chain reaction were used to sensitively quantify myeloma cells in bone marrow and peripheral blood stem cell collections, taken at a similar time, from eight patients with multiple myeloma. Levels of residual disease in the peripheral blood stem cell harvests were variable and did not reflect the tumour burden in the marrow. Peripheral blood stem cells contained 1.7 to 23 700‐fold fewer myeloma cells compared with the bone marrow and would have resulted in reinfusion of 0.08 to 59 480‐fold fewer myeloma cells based on total reinfused CFU‐GM and 0.24 to 24 700‐fold fewer myeloma cells based on total reinfused nucleated cells. Assuming that the proportion of clonogenic myeloma cells is equivalent, peripheral blood stem cells may be better than bone marrow as a source of haemopoietic stem cells for transplantation in multiple myeloma. The clinical follow‐up suggested that patients transplanted with peripheral blood stem cells containing a low number of myeloma cells had better disease control than those transplanted with peripheral blood stem cells containing a high number.

An investigation of admixture in an Australian Aboriginal Y-chromosome STR database

Y-chromosome specific STR profiling is increasingly used in forensic casework. However, the strong geographic clustering of Y haplogroups can lead to large differences in Y-STR haplotype frequencies between different ethnicities, which may have an impact on database composition in admixed populations. Aboriginal people have inhabited Australia for over 40,000 years and until ∼300 years ago they lived in almost complete isolation. Since the late 18th century Australia has experienced massive immigration, mainly from Europe, although in recent times from more widespread origins. This colonisation resulted in highly asymmetrical admixture between the immigrants and the indigenes. A State jurisdiction within Australia has created an Aboriginal Y-STR database in which assignment of ethnicity was by self-declaration. This criterion means that some males who identify culturally as members of a particular ethnic group may have a Y haplogroup characteristic of another ethnic group, as a result of admixture in their paternal line. As this may be frequent in Australia, an examination of the extent of genetic admixture within the database was performed. A Y haplogroup predictor program was first used to identify Y haplotypes that could be assigned to a European haplogroup. Of the 757 males (589 unique haplotypes), 445 (58.8%) were identified as European (354 haplotypes). The 312 non-assigned males (235 haplotypes) were then typed, in a hierarchical fashion, with a Y-SNP panel that detected the major Y haplogroups, C-S, as well as the Aboriginal subgroup of C, C4. Among these 96 males were found to have non-Aboriginal haplogroups. In total, ∼70% of Y chromosomes in the Aboriginal database could be classed as non-indigenous, with only 169 (129 unique haplotypes) or 22% of the total being associated with haplogroups denoting Aboriginal ancestry, C4 and K* or more correctly K(xL,M,N,O,P,Q,R,S). The relative frequencies of these indigenous haplogroups in South Australia (S.A.) were significantly different to those seen in samples from the Northern Territory and Western Australia. In S.A., K* (∼60%) has a much higher frequency than C4 (∼40%), and the subgroup of C4, C4(DYS390.1del), comprised only 17%. Clearly admixture in the paternal line is at high levels among males who identify themselves as Australian Aboriginals and this knowledge may have implications for the compilation and use of Y-STR databases in frequency estimates.

Molecular identification of python species: Development and validation of a novel assay for forensic investigations

Python snake species are often encountered in illegal activities and the question of species identity can be pertinent to such criminal investigations. Morphological identification of species of pythons can be confounded by many issues and molecular examination by DNA analysis can provide an alternative and objective means of identification. Our paper reports on the development and validation of a PCR primer pair that amplifies a segment of the mitochondrial cytochrome b gene that has been suggested previously as a good candidate locus for differentiating python species. We used this DNA region to perform species identification of pythons, even when the template DNA was of poor quality, as might be the case with forensic evidentiary items. Validation tests are presented to demonstrate the characteristics of the assay. Tests involved the cross-species amplification of this marker in non-target species, minimum amount of DNA template required, effects of degradation on product amplification and a blind trial to simulate a casework scenario that provided 100% correct identity. Our results demonstrate that this assay performs reliably and robustly on pythons and can be applied directly to forensic investigations where the presence of a species of python is in question.

Haplotype data for 16 Y-chromosome STR loci in Aboriginal and Caucasian populations in South Australia.

UNLABELLED Y-STR haplotype data was obtained using the AmpFlSTR(®) YFiler™ PCR Amplification Kit (Applied Biosystems, Foster City, CA) for 1079 Caucasian and 766 Australian Aboriginal individuals. Haplotype diversity was similar in both populations, however discrimination capacity was higher in Caucasians than Aborigines (0.946 compared to 0.692). Locus DYS385, which was considered as a single locus, was the most diverse marker in both populations (0.836 in Caucasians and 0.905 in Aborigines). POPULATION The South Australian Aboriginal and Caucasian databases were compiled from casework reference profile information held on the South Australian Criminal, Reference and Evidence DNA Database (SACREDD). Ethnicity was assigned based on self-declaration.

Purging of myeloma cells using all-trans retinoic acid in a mouse model

OBJECTIVE The 5T33 murine model of multiple myeloma was used to investigate the potential of all-trans retinoic acid (ATRA) to purge clonogenic myeloma cells from autologous hemopoietic stem-cell harvests by differentiating immature 5T33 cells into terminal-stage plasma cells with limited repopulation capacity. MATERIALS AND METHODS 5T33 cells were treated with 10 microM ATRA and the effect on cell clonogenicity was determined by measuring the time to paraprotein detection in C57Bl/KaLwRij mice compared to control animals. Cell differentiation and apoptosis following ATRA treatment were investigated using flow cytometry and caspase-3 assay. Treatment with ATRA resulted in a 33% reduction in the in vitro cloning efficiency of 5T33 cells. Reduced in vitro clonogenicity of 5T33 cells following ATRA treatment was supported by a 16-49% increase in the time taken for C57Bl/KaLwRij mice to develop paraprotein following injection of 5T33 cells pretreated with ATRA for 8 days. Although ATRA was shown not to alter the in vitro growth characteristics of 5T33 cells, significant inhibition of apoptosis was observed. RESULTS Treatment with ATRA also resulted in an increase in the proportion of 5T33 cells expressing the CD54 adhesion molecule, which is known to be highly expressed on mature myeloma cells. CONCLUSION The ability of ATRA to decrease the clonogenicity of 5T33 cells in vitro and increase the time to disease development in vivo suggests that this drug may be useful for purging autologous stem cell harvests in the clinical setting.

A mass spectrometry-based forensic toolbox for imaging and detecting biological fluid evidence in finger marks and fingernail scrapings

During a crime, biological material such as blood or vaginal fluid may become smeared on the fingers of the victim or suspect or trapped under their fingernails. The type of trapped fluid is extremely valuable forensic information. Furthermore, if either person touches an object at the crime scene with their ‘contaminated’ finger then a ‘contaminated’ finger mark may be deposited. Such marks have great value as they could identify not only who deposited the mark but also who they touched and which part of the body they touched. Here, we describe preliminary work towards a ‘toolbox’ of techniques based on mass spectrometry (MS) for the identification of biological fluid traces under fingernails or the imaging of them in finger marks. Liquid chromatography-multidimensional MS was effective for the detection of protein biomarkers characteristic of vaginal fluid and blood trapped under fingernails, even after hands had been washed. In regard to examination of finger marks for the presence of biological fluids, the most practical implementation of any technique is to integrate it with, but after, routine crime scene finger mark enhancement has been applied. Here, we demonstrate the usage of matrix-assisted laser desorption ionization-time of flight-MS for the detection and mapping of proteins and peptides from body fluids in finger marks, including marks enhanced using aluminium-containing magnetic powder and then ‘lifted’ with adhesive tape. Hitherto, only small molecules have been detected in enhanced, lifted marks. In a novel development, aluminium in the enhancement powder assisted ionization of small molecules in finger marks to the extent that conventional matrix was not required for MS.

South Australian Aboriginal sub-population data for the nine AMPFlSTR® Profiler Plus™ short tandem repeat (STR) loci

The weighting of DNA evidence can be estimated using various models. Each model relies on allele frequencies, which are obtained from data accumulated in a population database. This report analyses sub-population data for 325 Aboriginal Australian residents of South Australia at nine autosomal Profiler Plus short tandem repeat (STR) loci.

Species identification of protected carpet pythons suitable for degraded forensic samples

In this paper we report on the identification of a section of mitochondrial DNA that can be used to identify the species of protected and illegally traded pythons of the genus Morelia. Successful enforcement of wildlife laws requires forensic tests that can identify the species nominated in the relevant legislation. The potentially degraded state of evidentiary samples requires that forensic investigation using molecular genetic species identification is optimized to interrogate small fragments of DNA. DNA was isolated from 35 samples of Morelia spilota from which the complete cytochrome b was sequenced. The ND6 gene was also sequenced in 32 of these samples. Additional DNA sequences were generated from 9 additional species of Morelia. The sequences were aligned by Geneious and imported into MEGA to create phylogenetic trees based on the entire complex of approximately 1,706 base pairs (bp). To mimic degraded DNA, which is usually found in forensic cases, short sub-sections of the full alignment were used to generate phylogenetic trees. The sub-sections that had the greatest DNA sequence information were in parts of the cytochrome b gene. Our results highlight that legislation is presently informed by inadequate taxonomy. We demonstrated that a 278 bp region of the cytochrome b gene recovered the topology of the phylogenetic tree found with the entire gene sequence and correctly identified species of Morelia with a high degree of confidence. The locus described in this report will assist in the successful prosecution of alleged illegal trade in python species.

Characterisation of novel and rare Y-chromosome short tandem repeat alleles in self-declared South Australian Aboriginal database

Y-chromosome short tandem repeats (Y-STRs) are used in forensic science laboratories all over the world, as their application is wide and often vital in solving casework. Analysis of an in-house database of South Australian self-declared Aboriginal males held by Forensic Science South Australia (FSSA) using the Applied Biosystem’s AmpFℓSTR® Yfiler™ PCR Amplification Kit revealed 43 variant Y-STR alleles at 6 of the 17 loci. All variant alleles were sequenced to determine the exact repeat structure for each. As a high level of admixture has previously been found within the SA Aboriginal database, samples were haplogrouped using Y-SNPs to determine their likely geographical origin. Although a number of variant alleles were associated with non-Aboriginal Y-haplogroups, a high frequency was observed within the Australian K-M9 lineage. Detailed knowledge of these variant alleles may have further application in the development of new DNA markers for identification purposes, and in population and evolutionary studies of Australian Aborigines.