DNA extraction for short tandem repeat typing from mixed samples using anti-human leukocyte CD45 and ABO blood group antibodies

doi:10.1016/j.fsigen.2014.01.001

Forensic Science International: Genetics

Volume 10, May 2014, Pages 17-22

https://doi.org/10.1016/j.fsigen.2014.01.001 Get rights and content

Abstract

DNA testing from mixed cell samples can be difficult to use successfully in criminal investigations. Here, we present a method for the extraction of DNA from mixed bloodstains involving plural contributors, after antibody-microbead captured cell separation. This method, together with the multiplex short tandem repeat typing presented, has proven highly successful in the recovery of DNA profiles corresponding to the ABO blood type. Methodological steps include magnetic separation using leukocyte specific CD45 antibody-coated microbeads and centrifugal separation of leukocyte agglutination by ABO antibody. The detection results of variable mixed ratio showed that the target DNA was detected accurately as low as 1:512 mixed ratio, regardless of the large amount of the background DNA present. The method presented here is applicable to PCR-based identification for various kinds of mixed samples.

Introduction

Biological samples from a crime scene often contain a mixture of various cells from more than one person. For example, clothing which a blood soaked into may also contain epithelial cells from the wearer and a secretion from assailant. Blood obtained from a door handle or a knob may have been handled by several people. Thus, there are many situations when various kind of biological material can be collected.

A mixture in a DNA profile can be recognized by the presence of more than two alleles at any locus within the profile. There will be several loci that have three or four alleles present and a loss of peak balance. However, it is sometimes difficult to ascertain whether it is really a mixed sample or the artifacts in the profile such as stutter peak.

Having determined that the profile is really mixed, the first problematic task is to determine how many contributors are presented in the profile. Two person mixtures are most commonly seen in forensic casework, and can be distinguished as such by a maximum of four bands present at any locus. In a two-person mixture case when we can discriminate major and minor profiles, the interpretation becomes more difficult where the minor profile is less than one-third of the height of the major profile, because we cannot distinguish true allele from stutter peak [1]. However, if all peak heights exhibit almost same level, four bands can be interpreted as six different combinations. In practice, we do not easily know how many contributors typically contribute because of allele drop-out or drop-in, which can sometimes occur in LCN samples [2], [3], [4], [5]. Therefore, a separative extraction of DNA using cell-specific antibody would be a substantial improvement for DNA typing from mixed sample.

Section snippets

Mixed stain preparations

As a test case, we used a mixed whole blood sample containing an equal contribution from three men of different blood types (A, B and O) in a test tube (including EDTA). The ABO blood types and DNA types of the three men were previously determined. One mL of mixed blood was dropped into four places on cotton cloth and dried at room temperature for 3 h. Each stain in the cloth was cut into four pieces (approximately 0.5 cm × 0.5 cm) with sterile scissors and each piece transferred to one of four 15 mL

Separation of specific leukocytes

A representative result of the collection of leukocytes with A-antigen is shown in Fig. 1. Leukocytes expressing A-antigen were collected by leukocyte specific antibody CD45, and followed by an anti-A antibody (Pellet A) addition. Then, the pellet was transferred to a slide glass and stained with hematoxylin and eosin. We observed the agglutination of leukocytes clearly by administration of the specific antibody.

STR typing

DNA typing using the PowerPlex® Y23 Kit System are shown in Fig. 2 and Identifiler®

Discussion

Mixtures of DNA from two or more individuals are common in some forensic cases and the interpretation of the DNA profiles must be performed carefully [10], [11]. In evaluating the evidence, an analyst must decide whether the DNA source in the sample of interest is from a single individual or more than one person [12]. This may be accomplished by examining the number of alleles detected at each locus as well as identifying severely imbalanced peak height ratios, or pronounced peaks in the

References (18)

T.M. Clayton et al.
Analysis and interpretation of mixed forensic stains using DNA STR profiling
Forensic Sci. Int.
(1998)
J.S. Buckleton et al.
Towards understanding the effect of uncertainty in the number of contributors to DNA stains
Forensic Sci. Int. Genet.
(2007)
P. Gill et al.
DNA commission of the International Society of Forensic Genetics: recommendations on the evaluation of STR typing results that may include drop-out and/or drop-in using probabilistic methods
Forensic. Sci. Int. Genet.
(2012)
B. Falini et al.
Variable expression of leucocyte-common (CD45) antigen in CD30 (Ki1)-positive anaplastic large-cell lymphoma: implications for the differential diagnosis between lymphoid and nonlymphoid malignancies
Hum. Pathol.
(1990)
H. Haned et al.
Exploratory data analysis for the interpretation of low template DNA mixtures
Forensic Sci. Int. Genet.
(2012)
P. Gill et al.
A new methodological framework to interpret complex DNA profiles using likelihood ratios
Forensic Sci. Int. Genet.
(2013)
J.M. Butler
Forensic challenges: degraded DNA, mixtures, and LCN
K. Honda et al.
Selective blood-DNA extraction from mixed stain using ABO antibody for short tandem repeat typing
Forensic Sci. Int. Genet. Suppl.
(2013)
P. Gil et al.
DNA commission of the International Society of Forensic Genetics: recommendations on the interpretation of mixtures
Forensic Sci. Int.
(2006)

There are more references available in the full text version of this article.

Cited by (12)

A preliminary study on identification of the blood donor in a body fluid mixture using a novel compound genetic marker blood-specific methylation-microhaplotype
2024, Forensic Science International: Genetics
Blood-containing mixtures are frequently encountered at crime scenes involving violence and murder. However, the presence of blood, and the association of blood with a specific donor within these mixtures present significant challenges in forensic analysis. In light of these challenges, this study sought to address these issues by leveraging blood-specific methylation sites and closely linked microhaplotype sites, proposing a novel composite genetic marker known as “blood-specific methylation-microhaplotype”. This marker was designed to the detection of blood and the determination of blood donor within blood-containing mixtures. According to the selection criteria mentioned in the Materials and Methods section, we selected 10 blood-specific methylation-microhaplotype loci for inclusion in this study. Among these loci, eight exhibited blood-specific hypomethylation, while the remaining two displayed blood-specific hypermethylation. Based on data obtained from 124 individual samples in our study, the combined discrimination power (CPD) of these 10 successfully sequenced loci was 0.999999298. The sample allele methylation rate (Ram) was obtained from massive parallel sequencing (MPS), which was defined as the proportion of methylated reads to the total clustered reads that were genotyped to a specific allele. To develop an allele type classification model capable of identifying the presence of blood and the blood donor, we used the Random Forest algorithm. This model was trained and evaluated using the Ram distribution of individual samples and the Ram distribution of simulated shared alleles. Subsequently, we applied the developed allele type classification model to predict alleles within actual mixtures, trying to exclude non-blood-specific alleles, ultimately allowing us to identify the presence of blood and the blood donor in the blood-containing mixtures. Our findings demonstrate that these blood-specific methylation-microhaplotype loci have the capability to not only detect the presence of blood but also accurately associate blood with the true donor in blood-containing mixtures with the mixing ratios of 1:29, 1:19, 1:9, 1:4, 1:2, 2:1, 7:1, 8:1, 31:1 and 36:1 (blood:non-blood) by DNA mixture interpretation methods. In addition, the presence of blood and the true blood donor could be identified in a mixture containing four body fluids (blood:vaginal fluid:semen:saliva = 1:1:1:1). It is important to note that while these loci exhibit great potential, the impact of allele dropouts and alleles misidentification must be considered when interpreting the results. This is a preliminary study utilising blood-specific methylation-microhaplotype as a complementary tool to other well-established genetic markers (STR, SNP, microhaplotype, etc.) for the analysis in blood-containing mixtures.
Single cell genomics applications in forensic science: Current state and future directions
2023, iScience
Standard methods of mixture analysis involve subjecting a dried crime scene sample to a “bulk” DNA extraction method such that the resulting isolate compromises a homogenized DNA mixture from the individual donors. If, however, instead of bulk DNA extraction, a sufficient number of individual cells from the mixed stain are subsampled prior to genetic analysis then it should be possible to recover highly probative single source, non-mixed scDNA profiles from each of the donors. This approach can detect low DNA level minor donors to a mixture that otherwise would not be identified using standard methods and can also resolve rare mixtures comprising first degree relatives and thereby also prevent the false inclusion of non-donor relatives. This literature landscape review and associated commentary reports on the history and increasing interest in current and potential future applications of scDNA in forensic genomics, and critically evaluates opportunities and impediments to further progress.
Recovery of single source DNA profiles from mixtures by direct single cell subsampling and simplified micromanipulation
2021, Science and Justice
Citation Excerpt :
Some of these techniques rely on the binding of antibodies (Ab) to cellular antigens in a cell type specific manner (e.g. white blood cells (CD45 [4,12]), epithelial cells (CD227 [18], cytokeratin [15]) or via allele specific interaction with polymorphic blood groups [4] or MHC [13,17] markers. After Ab binding, cell separation takes place by fluorescence assisted cell sorting FACS [13,17,18], microbead separation followed by centrifugation of allele specific agglutinates [4] or digital cell sorting [12,15]. These methods, with the exception of digital cell sorting of individual fluorescent antibody coated cells, produce bulk enrichment of the cellular fractions and show promise for future routine use after further developmental work.
Deconvolution of forensic DNA mixtures into their individual component DNA (geno)types is of great investigative value, though often complex and difficult. Two-person mixtures comprising a major and minor contributor are often easily interpreted although, when the DNA ratio of the two individuals is approximately equal (~1:1), deconvolution and interpretation becomes much more difficult. To address this issue, a physical separation of individual-, two- or three- cell subsamples prior to autosomal STR analysis was performed using a simplified micromanipulation technique paired with a decreased reaction volume and increased cycle number PCR. Using this method, single and multiple buccal epithelial cells were collected from a 1:1 two-person mixture (i.e. from individual 'A' and 'B') and directly amplified, omitting standard DNA extraction and purification steps. Single cell subsamples resulted in partial single-source profiles for both contributors while, in accordance with expectations of a quasi-binomial sampling schema, two- and three-cell subsamples resulted in single source informative partial profiles of individual A and individual B as well as complete consensus profiles, and equally mixed 1:1 (2-cell subsamples) and 2:1 (3-cell subsamples) admixed profiles of individual A and B. This proof-of-concept approach shows promise in permitting the DNA deconvolution of mixed samples where the individual contributors are present in similar amounts that would otherwise be difficult to interpret, resulting in an increase in evidentiary value. The subsampling approach can be readily investigated for DNA casework applications without additional investment in costly, new equipment, requiring only a stereo microscope and a tungsten needle.
Purification and concentration of DNA using L-fucose-specific lectin
2017, Forensic Science International: Genetics Supplement Series
Citation Excerpt :
However, contamination or mixed cell samples cause further difficulties for DNA testing and interpretation. Therefore, we previously developed a selective extraction method using cell agglutination materials [1]. Lectins are classified into a small number of specificity groups according to their type of blood precipitation.
Lectins are classified into a small number of specificity groups according to their binding affinity to carbohydrate polymers. Therefore, we speculated that l-fucose lectin can combine with 5-carboxy-deoxyribose (pentose) instead of l-fucose (hexose), because of the similarity of chemical structure.
Based on this supposition, we successfully purified and concentrated DNA using l-fucose lectin, which can cause agglutination of DNA from hemolytic bloodstains. This result indicates that lectin treatment could be an additional method used for purification of extracted DNA or degraded blood samples. The binding of l-fucose-specific lectin to deoxyribose allows the specific collection of DNA from the sediment of lectin agglutination. Therefore we concluded that, for old samples with a cell membrane that has been destroyed, we may collect the isolated bare DNA more effectively by adding lectin.
DNA analysis overturns the death sentence of a condemned criminal held in custody for 48 years
2015, Forensic Science International: Genetics
FACS separation of non-compromised forensically relevant biological mixtures
2015, Forensic Science International: Genetics
Citation Excerpt :
No studies have been published relating to mixed samples not containing sperm. As a wide variety of antibody markers are commercially available, choices which reflect cell types of interest, e.g. anti-CD45 (a white blood cell surface antigen [17] as used by Yano et al. [13]) and anti-CD227 (a mucosal epithelial cell surface antigen [18]), can be readily made. Since Schoell et al. [14,15], technological advancement has led to improvements in cell sorting machines [19–21], meaning that highly sensitive and fast cell sorting, down to the single-cell level, can now be achieved [21].
Although focusing attention on the statistical analysis of complex mixture profiles is important, the forensic science community will also benefit from directing research to improving the reduction of the incidence of mixtures before DNA extraction. This preliminary study analysed the use of fluorescence assisted cell sorting (FACS) for separation of cellular mixtures before DNA extraction, specifically mixtures of relatively fresh blood and saliva from two donors, prepared in 14 different mixture ratios. Improvements in the number of detectable alleles from the targeted cell type and overall profile quality were seen when compared to the results from unseparated samples. STRmix calculations revealed increases in likelihood ratios after separation, demonstrating the potential for higher probative values to be obtained from forensically relevant mixtures after subjecting them to FACS than from unsorted samples.

View all citing articles on Scopus

View full text

Short CommunicationDNA extraction for short tandem repeat typing from mixed samples using anti-human leukocyte CD45 and ABO blood group antibodies

Abstract

Introduction

Section snippets

Mixed stain preparations

Separation of specific leukocytes

STR typing

Discussion

Forensic Sci. Int.

Forensic Sci. Int. Genet.

Forensic. Sci. Int. Genet.

Hum. Pathol.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet. Suppl.

DNA commission of the International Society of Forensic Genetics: recommendations on the interpretation of mixtures

Forensic Sci. Int.

Short Communication
DNA extraction for short tandem repeat typing from mixed samples using anti-human leukocyte CD45 and ABO blood group antibodies