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«Thesis ANALYTICAL FIGURES OF MERIT AS A MEANS TO COMPARE METHODS: AN EXAMPLE USING LATERAL FLOW IMMUNOCHROMATOGRAPHIC TEST STRIPS AND REAL-TIME PCR ...»

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BOSTON UNIVERSITY

SCHOOL OF MEDICINE

Thesis

ANALYTICAL FIGURES OF MERIT AS A MEANS TO COMPARE METHODS:

AN EXAMPLE USING LATERAL FLOW IMMUNOCHROMATOGRAPHIC

TEST STRIPS AND REAL-TIME PCR

by

CRYSTAL MAE (SIMSON) OECHSLE

B.S., Ohio University, 2007 Submitted in partial fulfillment of the requirements for the degree of Master of Science Approved By First Reader ________________________________________________

Catherine M. Grgicak, Ph.D.

Instructor Biomedical Forensic Sciences Second Reader ________________________________________________

Amy L. Barber, M.S.

DNA Unit Supervisor & Acting Technical Leader Massachusetts State Police Crime Laboratory Acknowledgements I would like to express my sincere gratitude to all those individuals who provided me with their reassurance, guidance, and expertise throughout the course of this project. Without their support, this thesis would not have come to fruition. I would specifically like to thank my husband, Stephan, for his endless encouragement; my co-workers at the Massachusetts State Police, including Amy Barber, Sandra Haddad, Joanne Sgueglia, Kristen Sullivan, and Maureen McCabe, for their commitment to excellence in the field of forensics; and my advisor at Boston University, Catherine Grgicak, for her time, patience, and direction.

iii

ANALYTICAL FIGURES OF MERIT AS A MEANS TO COMPARE METHODS:

AN EXAMPLE USING LATERAL FLOW IMMUNOCHROMATOGRAPHIC

TEST STRIPS AND REAL-TIME PCR

CRYSTAL MAE (SIMSON) OECHSLE

Boston University School of Medicine, 2011 Major Professor: Catherine M. Grgicak, Ph. D., Instructor Biomedical Forensic Sciences

ABSTRACT

Forensic evidence samples are often subjected to a variety of biological fluid identification methods before potentially probative items are forwarded to DNA analysis. Serologically based screening techniques may provide valuable insight in how to proceed with a specific sample; however, they consume a vital portion of the available evidence. One limitation of these techniques is that they are qualitative or semi-quantitative in nature, relying on visual interpretation by analysts. In contrast, recent advances in real-time Polymerase Chain Reaction (PCR) quantification allow for the simultaneous detection of total human and male DNA. Commercially available quantification kits do not identify bodily fluids, per se, but do yield information regarding the proportion of the DNA present in the sample and the downstream amplifiability of the genetic material. Therefore, real-time quantification could theoretically be used as a screening method for subsequent PCR amplification of human specific Short Tandem Repeat (STR)

–  –  –

genetic material, is probative.

In this study, dilution series of either semen or male saliva were prepared in either buffer or female blood. Semen and saliva dilution samples were subjected to lateral flow immunochromatographic test strips for the detection of either semenogelin or salivary -amylase, respectively. All samples were also subjected to real-time PCR analysis, which allowed for simultaneous quantification of total human and male DNA. Because the chemistries and signal outputs of each method differs, a direct comparison of signal could not be made.

Instead, analytical figures of merit based on the volume of bodily fluid were evaluated. With the exception of the semen dilution series analyzed on the immunoassay cards, which displayed evidence of the high-dose hook effect, loglinear relationships between signal and volume were observed for both platforms.

Utilizing this relationship and the theory of the propagation of random errors the Limits of Detection (LOD) were determined to be 0.05 L of saliva for the RSID™ Saliva cards, 0.03 L saliva for Quantifiler® Duo, and 0.001 L of semen for Quantifiler® Duo. Due to its stability in various matrices, sensitivity, low limits of detection, and reproducibility, quantitative PCR is a viable and effective screening method for subsequent DNA profiling.

–  –  –

Table 1. List and preparation of the 11 samples in each 14 body fluid dilution series.

Values in parenthesis represent dilutions of the body fluid of interest prior to its addition to the mixture.

Table 2. Immunoassay card MBS and standard deviation 30 values used to calculate MDS values (shown in bold), which are plotted as dashed horizontal lines in Figures 9A (saliva-blood and saliva-TE) and 9B (semen-blood and semen-TE) and later in Figures 11A (saliva) and 11B (semen).

Table 3. Calibration sensitivities for body fluid dilution series 34 analyzed with RSID™ Saliva cards, RSID™ Semen cards, and Quantifiler® Duo.

Table 4. Analytical sensitivities for Saliva-Blood dilution series 35 analyzed with RSID™ Saliva cards and Quantifiler® Duo.





Table 5. Analytical sensitivities for Saliva-TE dilution series 35 analyzed with RSID™ Saliva cards and Quantifiler® Duo.

Table 6. Analytical sensitivities for Semen-Blood dilution series 36 analyzed with RSID™ Semen cards and Quantifiler® Duo.

Table 7. Analytical sensitivities for Semen-TE dilution series 36 analyzed with RSID™ Semen cards and Quantifiler® Duo.

Table 8. Overall calibration sensitivities for RSID™ Saliva 39 cards, RSID™ Semen cards, and Quantifiler® Duo.

Table 9. Presence of signal – either a red band in the test 41 region for immunoassay cards or a CT value for real-time PCR – in detection of Saliva.

Table 10. Presence of signal – a red band in the test region 42 for immunoassay cards, sperm on microscope slides, or a CT value for real-time PCR – in detection of Semen.

Table 11. Limits of Detection of saliva and semen for the 46 various detection methods.

–  –  –

Figure 1. Illustration of the principle behind the production 6 of a red-band in the test region of immunoassay cards using colloidal gold labeled antibodies.

Figure 2. Illustration of lateral flow immunochromatographic 6 test strip mechanism.

Figure 3. Illustration of the three phases of real-time PCR: 8 exponential, linear, and plateau.

The Cycle Threshold (CT), or the cycle number at which fluorescence crosses a set threshold, is also depicted.

Figure 4. Illustration of the Taqman® probe detection method 11 of real-time PCR.

Figure 5. Flow chart of sample preparation for body fluid 15 mixtures.

Figure 6. (A) Third replicate of the saliva-TE dilution series 23 run on RSID™ Saliva lateral flow immunochromatographic test strips.

(B) Third replicate of the semen-blood dilution series run on RSID™ Semen lateral flow immunochromatographic test strips.

Figure 7. ImageJ software output for the third replicate of 24 the semen-blood 1:50 dilution (immunoassay card seen in Figure 6).

Figure 8. Real-time PCR amplification plots for the third 27 replicate of the semen-blood dilution series.

The horizontal green line represents the signal intensity threshold needed for the CT to be recorded.

Figure 9. Sensitivity curves for immunochromatographic 29 test strips and real-time PCR quantification platforms.

(A) Saliva-Blood and Saliva-TE dilution series on RSID™ Saliva cards, (B) Semen-Blood and Semen-TE dilution series on RSID™ Semen cards, (C) Saliva-Blood and Saliva-TE dilution series with Quantifiler® Duo, and (D) Semen-Blood and Semen-TE dilution series with Quantifiler® Duo.

–  –  –

Figure 11. RSID™ immunoassay card calibration sensitivity 39 plotted against Quantifiler® Duo calibration sensitivity.

(A) Overall Saliva and (B) Overall Semen.

Figure 12. RSID™ immunoassay card RSDs plotted against 52 Quantifiler® Duo RSDs.

(A) Overall Saliva and (B) Overall Semen.

–  –  –

CCD Charge Coupled Device CODIS Combined DNA Index System DNA Deoxyribonucleic Acid DTT Dithiothreitol FBI Federal Bureau of Investigation

–  –  –

FRET Fluorescent Resonance Energy Transfer HPV Human Papillomavirus IgG Immunoglobulin G IUPAC International Union of Pure and Applied Chemistry JPEG Joint Photographic Experts Group image

–  –  –

ProK Proteinase K qPCR Quantitative Polymerase Chain Reaction R2 Correlation Coefficient RNA Ribonucleic Acid RPM Revolutions per Minute RPPH1 Ribonuclease P RNA Component H1 RSD Relative Standard Deviation RSID Rapid Stain Identification

–  –  –

SRY Sex-determining Region Y STR Short Tandem Repeat SWGDAM Scientific Working Group on DNA Analysis Methods Taq Thermus aquaticus TE Tris-EDTA (Ethylenediamine Tetra-Acetic acid)

–  –  –

Forensic evidence samples are often subjected to a variety of biological fluid identification methods before potentially probative items are forwarded for DNA analysis. Serologically based screening techniques may provide valuable insight on how to proceed with a specific sample; however, they consume a significant portion of the available evidence. Another limitation of these techniques is that they are qualitative or semi-quantitative in nature, relying on visual interpretation by analysts. In contrast, recent advances in real-time PCR quantification chemistries allow for the simultaneous fluorescent detection of total human and male DNA. Where real-time PCR does not identify body fluids, per se, it can yield information on the proportion of the biological components present in the sample and the downstream amplifiability of the genetic material.

Therefore, real-time quantification could theoretically be used as a screening method for subsequent PCR amplification of human-specific STR DNA profiles, and due to Standard 9.4 of the FBI’s Quality Assurance Standards for Forensic DNA Testing Laboratories, real-time PCR is already validated for use by many forensic laboratories [1].

Real-time quantification with a dual human and male platform would be particularly valuable as a diagnostic approach in cases where development of a male DNA profile, especially in the abundance of female genetic material, is probative. Such a scenario often occurs with sexual assaults, which make up a significant portion of DNA backlogs. As an example, sexual assaults accounted for approximately 7% of all violent crime nationally in 2009 [2]. Evidence related to sexual assault cases poses a unique analytical challenge because of the vast number of scenarios encountered, which is one reason why biological screening methods are commonly utilized in forensic laboratories. At the Massachusetts State Police Crime Laboratory, for instance, the current protocol for processing sexual assault evidence collection kits involves extraction and microscopic examination for the presence of sperm cells, which is typically followed by semenogelin and/or amylase testing [3-7]. This is all before an item is even submitted for DNA analysis, which consists of its own separate extraction and quantification steps. It is recognized that such an approach is redundant, timely, and not cost effective; however, there is little available in the literature that directly compares the technologies to assess whether qPCR can effectively replace traditional screening methods. Any proposed procedure that would replace serology would need to be at least as sensitive, and have a comparable limit of detection. Sensitivity and limits of detection are two important analytical criteria in sexual assault evidence examination because, as previously mentioned, these cases often involve low levels of the male biological material of interest mixed with an overwhelming amount of female biological material. A high female to male ratio can occur for a number of reasons including digital penetration, lack of ejaculation, and the reportedly high frequency of azoospermia (i.e. the absence of spermatozoa in semen) in the general population [8].

The goal of the following set of experiments is to collect data that will support or refute a method for streamlining the processing of sexual assault evidence collection kits, where traditional evaluation of sperm cells and proteins is replaced with a human and male specific dual quantification method thus saving time, energy, and resources. In this study, multiple dilution series of either semen or male saliva were prepared in either TE buffer or female blood.

Semen and saliva dilution samples were subjected to RSID™ lateral flow immunochromatographic test strips for the detection of either semenogelin or salivary -amylase, respectively. Results were determined visually as well as through the use of ImageJ software, which is freeware available from the National Institute of Health (NIH) [9]. All dilution samples were also subjected to real-time PCR quantification with Quantifiler® Duo, a human and male multiplex quantification chemistry.

Immunochromatographic Test Strips The chemistries, analytes, and signal outputs of both immunochromatographic test strips and real-time PCR differ. To appreciate these differences, an understanding of the theory behind each is needed.



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