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Comparison and optimization of DNA recovery from sperm vs. epithelial cells using laser capture microdissection technology and an immunofluorescent staining system
Forensic Science International: Genetics Supplement Series 3 (2011) e224–e225
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Comparison and optimization of DNA recovery from sperm vs. epithelial cells using laser capture microdissection technology and an immunofluorescent staining system G. Axler-DiPerte *, S. Orans, A. Singh, T. Caragine, M. Prinz, Z.M. Budimlja New York City Office of Chief Medical Examiner, Department of Forensic Biology, 421 East 26th Street, New York, NY, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 August 2011 Accepted 31 August 2011
Laser capture microdissection (LCM) is an accurate and robust tool for isolation and separation of pure cell populations from heterogeneous mixtures via direct visualization and collection of elements. In this study, the capability of the P.A.L.M.1 LCM for separation of sperm and epithelial cells, combined with immunofluorescent Sperm HY-LITERTM stain was assessed. LCM was evaluated for its sensitivity, specificity, and reproducibility using sperm–epithelial mixture slides with varying sperm counts and ages ranging 1–9 years. In addition, slides stained with immunofluorescent dyes were tested. Separation of sperm and epithelial cells using LCM is a robust method for processing of mixed samples and generates interpretable single source DNA profiles. Immunofluorescent staining method is compatible with the platform. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Laser capture microdissection Immunofluorescence Sperm cell Epithelial cell Mixture Forensic
1. Introduction Mixtures of sperm and epithelial cells are commonly observed after sexual assaults and often generate mixed STR profiles following routine differential lysis DNA extraction. Complex mixture deductions can impact the statistical significance of the results, especially in cases of samples with low initial sperm amounts. When there is scarce material from the perpetrator due to biological or environmental factors, differential lysis separation can cause loss of material due to premature lysis of sperm cells. Here we describe our validation of P.A.L.M.1 LCM [1] for separation of Nuclear Fast Red (NFR) stained mixtures containing sperm and epithelial cells [2] for use in routine sexual assault casework. Our goal was to use LCM in the context of standard laboratory techniques with minimal modification, and to evaluate the immunofluorescent Sperm HY-LITERTM [3] stain as a method to increase sensitivity for future validation.
years) swabs stained with NFR + Sperm HY-LITERTM (Independent Forensics, Hillside, IL, USA). Sample separation was performed using the P.A.L.M.1 (Positioning and Ablation with Laser Microbeams) LCM System (Carl Zeiss MicroImaging, Bernried, Germany). Catapulting occurred into a 0.5-ml standard microcentrifuge tube mounted onto the collector with approximately 20 ml of extraction buffer inside the lid. DNA was extracted by heat-mediated lysis with Protienase K and DTT. Samples were quantified by Alu-based RT-PCR using Rotor-geneTM 6000 (Corbett Biosciences, QIAGEN, Du¨sseldorf, Germany), followed by 28 cycles of AmpF‘STR1 Identifiler1 (ABI, Foster City, CA, USA) DNA typing, capillary electrophoresis (ABI Prism1 3130xl Genetic Analyzer) and STR analysis (Genescan1 & Genotyper1).
3. Results and discussion 3.1. Sperm vs. epithelial cells
2. Material and methods Polyethylene-naphthalate (PEN) membrane slides were prepared from: single source sperm and epithelial cells; counted sperm: epithelial mixtures (1:1, 1:10, 1:50) stained with NFR; uncounted mixtures prepared from fresh and archived (up to 9
* Corresponding author. Tel.: +1 212 323 1497; fax: +1 212 323 1590. E-mail address: [email protected] (G. Axler-DiPerte). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.08.112
DNA yields from LCM catapulted samples varied widely from the theoretical amount expected (Fig. 1). Captures of 100 sperm cells (optimally 150–200) yielded 100% allele recovery across all replicates. Complete profiles were obtained for samples up to 8 years old. Sperm cell captures from either mixed source or single source slides gave higher DNA yields than all epithelial cell captures. A probable reason for this is non-specific pickup of adjacent sperm cells by static forces. Alternatively, defined epithelial cell capture counts provided less than the theoretical
G. Axler-DiPerte et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e224–e225
e225
HY-LITERTM staining was equivalent to the NFR processed slides (an average of 99% of alleles recovered). Under the FITC filter sperm cells can be easily distinguished, which can be useful for automated, software driven sperm searches. However, under the DAPI filter the pores of PEN membranes cause increased background fluorescence. 3.4. Technical concerns and recommendations
Fig. 1. DNA percent yield from LCM captures of (from left to right) 25, 50, 75, 100, 150, and 200 cells of each type. The average DNA yield is shown, with 100% representing the expected theoretical yield per cell number captured. Single source captures of sperm and epithelial cells are the product of 5 replicates, with the error bars showing the upper and lower yields. Captures from mixed source slides are the product of one capture.
DNA amount in all cases tested. This may be caused by the differing physical characteristics of this cell type. 3.2. Mixture separation In all cases where captures yielded sufficient DNA for analysis, profiles were either single source or the captured cell population was the major contributor. LCM was able to capture sufficient material to obtain STR profiles of both (sperm and epithelial cells) contributors, even in samples with a comparatively low sperm count (1:50). Epithelial cell captures often resulted in DNA contributions from the semen donors, despite attempts to exclude sperm cells from captured elements. This occurred most often in mixtures where sperm and epithelial cells were in near equal proportions, but also surprisingly in cases where sperm count was low. Most epithelial cell captures from mixed samples showed DNA contamination from the sperm donor, which can be explained by the fact that epithelial cells form aggregates on both the swab and slide, so that sperm cells may be enveloped in the aggregate and non-specifically co-selected. Five samples were tested using both, differential lysis DNA extraction and LCM. Results were comparable (data not shown). TM
3.3. Sperm HY-LITER
Sperm HY-LITERTM immunofluorescent staining performed well on previously NFR stained slides. DNA recovery after Sperm
One technical difficulty observed during the validation was incomplete capture of targeted cells or areas. This may be caused by several factors: the optimal laser settings can vary with ambient humidity and temperature; debris from swabs and stains transferred to the membrane can affect the laser’s ability to cut and propel a membrane segment; heat and chemical fixatives can cause local warping of the PEN-membrane and consecutively change the trajectory of the membrane fragments. Therefore, it is recommended that the laser settings are tested and optimized prior to any catapulting attempts to ensure complete captures. In addition, catapulting needs to be monitored and should not be automated as the local slide environment can require adjustment of the laser settings within the same slide. Multiple laser pulses should be used for the capture of larger areas. 4. Conclusions P.A.L.M.1 LCM can effectively separate the sperm component of mixtures from epithelial cells and debris. For standard STR typing 100–150 sperm cells are required and comparable results can be obtained using differential lysis. Since differential lysis is an inexpensive, and less labor intensive process, LCM should be reserved for evidence where differential lysis failed to produce a discernable male contributor profile. In addition, Sperm HYLITERTM immunofluorescent staining is compatible with this platform, while the potential of this method for automated sperm searches and downstream enhanced detection methods with Low Template (LT) DNA protocols need to be explored. Conflict of interest None. References [1] S. Seidl, et al., Contact-free isolation of sperm and epithelial cells by laser microdissection and pressure catapulting, Forensic Sci. Med. Pathol. (2) (2005) 153–157. [2] K. Elliott, et al., Use of laser microdissection greatly improves the recovery of DNA from sperm on microscope slides, Forensic Sci. Int. 137 (1) (2003) 28–36. [3] K.W.P. Miller, et al., Developmental validation of the SPERM HY-LITERTM kit for the identification of human spermatozoa in forensic samples, J. Forensic Sci. 56 (4) (2011) 853–865.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Comparison and optimization of DNA recovery from sperm vs. epithelial cells using laser capture microdissection technology and an immunofluorescent staining system G. Axler-DiPerte *, S. Orans, A. Singh, T. Caragine, M. Prinz, Z.M. Budimlja New York City Office of Chief Medical Examiner, Department of Forensic Biology, 421 East 26th Street, New York, NY, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 August 2011 Accepted 31 August 2011
Laser capture microdissection (LCM) is an accurate and robust tool for isolation and separation of pure cell populations from heterogeneous mixtures via direct visualization and collection of elements. In this study, the capability of the P.A.L.M.1 LCM for separation of sperm and epithelial cells, combined with immunofluorescent Sperm HY-LITERTM stain was assessed. LCM was evaluated for its sensitivity, specificity, and reproducibility using sperm–epithelial mixture slides with varying sperm counts and ages ranging 1–9 years. In addition, slides stained with immunofluorescent dyes were tested. Separation of sperm and epithelial cells using LCM is a robust method for processing of mixed samples and generates interpretable single source DNA profiles. Immunofluorescent staining method is compatible with the platform. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Laser capture microdissection Immunofluorescence Sperm cell Epithelial cell Mixture Forensic
1. Introduction Mixtures of sperm and epithelial cells are commonly observed after sexual assaults and often generate mixed STR profiles following routine differential lysis DNA extraction. Complex mixture deductions can impact the statistical significance of the results, especially in cases of samples with low initial sperm amounts. When there is scarce material from the perpetrator due to biological or environmental factors, differential lysis separation can cause loss of material due to premature lysis of sperm cells. Here we describe our validation of P.A.L.M.1 LCM [1] for separation of Nuclear Fast Red (NFR) stained mixtures containing sperm and epithelial cells [2] for use in routine sexual assault casework. Our goal was to use LCM in the context of standard laboratory techniques with minimal modification, and to evaluate the immunofluorescent Sperm HY-LITERTM [3] stain as a method to increase sensitivity for future validation.
years) swabs stained with NFR + Sperm HY-LITERTM (Independent Forensics, Hillside, IL, USA). Sample separation was performed using the P.A.L.M.1 (Positioning and Ablation with Laser Microbeams) LCM System (Carl Zeiss MicroImaging, Bernried, Germany). Catapulting occurred into a 0.5-ml standard microcentrifuge tube mounted onto the collector with approximately 20 ml of extraction buffer inside the lid. DNA was extracted by heat-mediated lysis with Protienase K and DTT. Samples were quantified by Alu-based RT-PCR using Rotor-geneTM 6000 (Corbett Biosciences, QIAGEN, Du¨sseldorf, Germany), followed by 28 cycles of AmpF‘STR1 Identifiler1 (ABI, Foster City, CA, USA) DNA typing, capillary electrophoresis (ABI Prism1 3130xl Genetic Analyzer) and STR analysis (Genescan1 & Genotyper1).
3. Results and discussion 3.1. Sperm vs. epithelial cells
2. Material and methods Polyethylene-naphthalate (PEN) membrane slides were prepared from: single source sperm and epithelial cells; counted sperm: epithelial mixtures (1:1, 1:10, 1:50) stained with NFR; uncounted mixtures prepared from fresh and archived (up to 9
* Corresponding author. Tel.: +1 212 323 1497; fax: +1 212 323 1590. E-mail address: [email protected] (G. Axler-DiPerte). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.08.112
DNA yields from LCM catapulted samples varied widely from the theoretical amount expected (Fig. 1). Captures of 100 sperm cells (optimally 150–200) yielded 100% allele recovery across all replicates. Complete profiles were obtained for samples up to 8 years old. Sperm cell captures from either mixed source or single source slides gave higher DNA yields than all epithelial cell captures. A probable reason for this is non-specific pickup of adjacent sperm cells by static forces. Alternatively, defined epithelial cell capture counts provided less than the theoretical
G. Axler-DiPerte et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e224–e225
e225
HY-LITERTM staining was equivalent to the NFR processed slides (an average of 99% of alleles recovered). Under the FITC filter sperm cells can be easily distinguished, which can be useful for automated, software driven sperm searches. However, under the DAPI filter the pores of PEN membranes cause increased background fluorescence. 3.4. Technical concerns and recommendations
Fig. 1. DNA percent yield from LCM captures of (from left to right) 25, 50, 75, 100, 150, and 200 cells of each type. The average DNA yield is shown, with 100% representing the expected theoretical yield per cell number captured. Single source captures of sperm and epithelial cells are the product of 5 replicates, with the error bars showing the upper and lower yields. Captures from mixed source slides are the product of one capture.
DNA amount in all cases tested. This may be caused by the differing physical characteristics of this cell type. 3.2. Mixture separation In all cases where captures yielded sufficient DNA for analysis, profiles were either single source or the captured cell population was the major contributor. LCM was able to capture sufficient material to obtain STR profiles of both (sperm and epithelial cells) contributors, even in samples with a comparatively low sperm count (1:50). Epithelial cell captures often resulted in DNA contributions from the semen donors, despite attempts to exclude sperm cells from captured elements. This occurred most often in mixtures where sperm and epithelial cells were in near equal proportions, but also surprisingly in cases where sperm count was low. Most epithelial cell captures from mixed samples showed DNA contamination from the sperm donor, which can be explained by the fact that epithelial cells form aggregates on both the swab and slide, so that sperm cells may be enveloped in the aggregate and non-specifically co-selected. Five samples were tested using both, differential lysis DNA extraction and LCM. Results were comparable (data not shown). TM
3.3. Sperm HY-LITER
Sperm HY-LITERTM immunofluorescent staining performed well on previously NFR stained slides. DNA recovery after Sperm
One technical difficulty observed during the validation was incomplete capture of targeted cells or areas. This may be caused by several factors: the optimal laser settings can vary with ambient humidity and temperature; debris from swabs and stains transferred to the membrane can affect the laser’s ability to cut and propel a membrane segment; heat and chemical fixatives can cause local warping of the PEN-membrane and consecutively change the trajectory of the membrane fragments. Therefore, it is recommended that the laser settings are tested and optimized prior to any catapulting attempts to ensure complete captures. In addition, catapulting needs to be monitored and should not be automated as the local slide environment can require adjustment of the laser settings within the same slide. Multiple laser pulses should be used for the capture of larger areas. 4. Conclusions P.A.L.M.1 LCM can effectively separate the sperm component of mixtures from epithelial cells and debris. For standard STR typing 100–150 sperm cells are required and comparable results can be obtained using differential lysis. Since differential lysis is an inexpensive, and less labor intensive process, LCM should be reserved for evidence where differential lysis failed to produce a discernable male contributor profile. In addition, Sperm HYLITERTM immunofluorescent staining is compatible with this platform, while the potential of this method for automated sperm searches and downstream enhanced detection methods with Low Template (LT) DNA protocols need to be explored. Conflict of interest None. References [1] S. Seidl, et al., Contact-free isolation of sperm and epithelial cells by laser microdissection and pressure catapulting, Forensic Sci. Med. Pathol. (2) (2005) 153–157. [2] K. Elliott, et al., Use of laser microdissection greatly improves the recovery of DNA from sperm on microscope slides, Forensic Sci. Int. 137 (1) (2003) 28–36. [3] K.W.P. Miller, et al., Developmental validation of the SPERM HY-LITERTM kit for the identification of human spermatozoa in forensic samples, J. Forensic Sci. 56 (4) (2011) 853–865.