Chapter 11: Serology Techniques: Past, Current, and Future

11.1: Introduction to Forensic Serology

• Forensic serology is the component of forensic biology that deals with the examination and identification of biological evidence.
• It focuses on determining the presence and identification of various bodily fluids such as blood, semen, and saliva in a questioned sample.

Class Characteristics and Individual Characteristics of Biological Evidence

• The identification of an unknown fluid sample is based on a comparison of the class characteristics of a sample with known standards of its class.
• Forensic identification typically involves bodily fluids, such as blood, semen, and saliva.
• If the presence of the bodily fluid is confirmed, the individual characteristics of the biological evidence are then determined to find out whether or not a bodily fluid sample has come from a particular individual.
• Short Tandem Repeats (STR): The most commonly utilized forensic DNA analysis used for human identification.
• Y-chromosomal STR analysis is often utilized for the investigation of sexual assault crimes.
• Mitochondrial DNA analysis is used for the identification of human remains.
• Establishing the probative value of a sample requires both the identification of its class characteristics and the individualization of its contributor.

Presumptive and Confirmatory Assays

• The identification of bodily fluids can be carried out using presumptive and confirmatory assays to identify the type of bodily fluid in question.
• Presumptive Assays   * Positive assay indicates the possibility of the presence of the bodily fluid in question.   * Negative assay indicates that the questioned bodily fluid is absent.
• Confirmatory Assays   * These assays are utilized to identify bodily fluids with higher certainty than presumptive assays.   * These are performed when a sample has to be identified as blood.

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Primary and Secondary Binding Assays

• Primary Binding Assays   * It involves the initial binding between a single epitope of an antigen and a single binding site of an antibody.   * They are very sensitive assays.
• Secondary Binding Assays   * They are less sensitive and easier to perform than primary binding assays.   * Precipitation-based assays have been used for species identification.   * Agglutination-based assays are more sensitive. It detects antigens located on the surface of cells or carriers, which are normally applied to blood group typing.

11.2: Primary Binding Assays

Enzyme-Linked Immunosorbent Assay (ELISA)

• It is an immuno-enzyme assay that can be used to detect and measure the antibody or antigen in question.
• Antibody-sandwich ELISA: The most common ELISA that is used in forensic serology.   * It is utilized to detect the prostate-specific antigen (PSA) to identify seminal stains and amylase for the identification of saliva.
• An antibody coating is formed by nonspecific adsorption onto a solid phase such as the wells of a polystyrene plate.
• Several enzymes such as alkaline phosphatase and horseradish peroxidase have been used as reporting enzymes to label the antiglobulin for ELISA.

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Immunochromatographic Assays

• The assay is carried out by loading a sample into the sample well.
• The antigen in the sample binds to the dye-labeled antibody already in the sample well to form an antigen–antibody complex.
• The complex then diffuses across the nitrocellulose membrane until it reaches the test zone.
• The antibody immobilized at the test zone traps the antigen–antibody complex to form an antibody–antigen–antibody sandwich.
• The presence of the antigen in the sample results in a colored vertical line at the test zone.
• The immunochromatographic device also utilizes a control zone to ensure that the device works properly and that the sample has diffused completely along the test strip.

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Common Immunochromatographic Assays for Forensic Applications

11.3: Secondary Binding Assays

Precipitation-Based Assays

• Immunodiffusion   * A passive method in which an antigen or an antibody or both are allowed to diffuse and therefore a gradient, from low to high concentration, is established for an antigen or an antibody or both.   * Single Immunodiffusion: A concentration gradient is established for either an antigen or an antibody.   * Double Immunodiffusion: A concentration gradient is established for both an antigen and an antibody.   * The Ouchterlony assay is named after the Swedish immunologist, Örjan Ouchterlony, who developed it. The assay can be performed in an agarose gel supported by a glass slide or polyester film

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• Immunoelectrophoretic Methods   * Immunoelectrophoresis (IEP): This technique uses electrophoresis to separate the antigen mixture before immunodiffusion.   * Crossed Immunoelectrophoresis (CRIE): Also known as two-dimensional IEP, is a modification of IEP.   * Rocket Immunoelectrophoresis: An antibody-containing agarose gel is used. The antigen is loaded into the well.   * Crossed-Over Immunoelectrophoresis: This technique is also known as counterimmunoelectrophoresis (CIE).

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Agglutination-Based Assays

• Agglutination reactions can be used as forensic serological assays such as for blood group typing and menstrual blood identification.
• Agglutination assays are qualitative, indicating the absence or presence of antigens or antibodies.
• Semi-quantitative assays results can be obtained by titration.
• Direct agglutination assays involve reactions in which an antibody interacts with antigens originally located on cell surfaces.
• In a hemagglutination reaction, an antibody binds to the antigens located on erythrocytes. This method is used for the identification of blood types.
• Agglutination inhibition assays: The presence of an antigen in question is indirectly detected.
• Passive agglutination assays: The antigen is coated on the surface of carrier cells such as tannic acid–treated sheep erythrocytes.

11.4: DNA Methylation Assays for Bodily Fluid Identification

• In the eukaryotic genome, methylation occurs at the cytosine residues commonly in the CpG dinucleotide sequences of both DNA strands.
• The “p” in CpG refers to the phosphodiester bond between cytosine and guanine.
• Cytosine methylation is carried out in vivo by a methyltransferase.
• Genomic Loci: Also known as tissue-specific differentially methylated regions (TDMRs).
• Methylation-sensitive restriction enzyme digestion polymerase chain reaction (MSRE-PCR) can be used for the rapid detection of DNA methylation.   * This method utilizes the methylation-sensitive restriction enzyme (MSRE).
• Bisulfite sequencing: The genomic DNA is treated with sodium bisulfite, which catalyzes the hydrolytic deamination of unmethylated cytosines.
• Methyl-DNA immunoprecipitation (MeDIP) is a useful method for isolating methylated DNA.

11.5: Forensic Applications of RNA-Based Assays and RNA Profiling

Messenger RNA-Based Assays

• mRNA-based Assays can potentially be used for wound age estimation and the age of biological stains.
• The tissue-specific genes that are utilized for bodily fluid identification.
• Reference genes: Constitutively expressed housekeeping genes are utilized as internal controls.

MicroRNA-Based Assays

• The biological function of microRNAs (miRNAs) is to regulate gene expression.
• The mature miRNA strand associated with the RNA-induced silencing complex (RISC) binds to its target mRNA.
• A single miRNA can bind different mRNA transcripts encoded by multiple genes.
• Animal miRNAs usually form a base pairing with their target mRNAs through partial complementary sequences, which lead to translation repression to inhibit protein synthesis.
• If the base pairing is exactly complementary to its target mRNA sequence, then the cleavage and the degradation of the target mRNA occur.

11.6: Proteomic Approaches Using Mass Spectrometry for Bodily Fluid Identification

• Mass spectrometry (MS) is a susceptible and rapid technique for protein identification from complex biological samples.

Mass Spectrometric Instrumentation for Protein Analysis

• Mass spectrometer: An analytical technique to identify a molecule by measuring the ratio of the mass (m) to the charge (z) of a charged molecule.   * A typical mass spectrometer instrument analyzes ionized molecules while in their gas phase.
• Ion source: Converts analytes into gaseous phase ions through ionization.
• Ionization: A process used by gaining a positive or a negative charge from a neutral species.
• Mass Analyzer: A part of a mass spectrometer where ions are accelerated under electric fields and separated based on their m/z ratio.
• Biomarker proteins can be identified by peptide sequencing using the tandem mass spectrometry (MS/MS) mode of operation in which a mass spectrometer uses two or more mass analyzers.
• After an initial mass analysis, individual peptide ions in the mass spectrum can be selected. The selected ions are known as precursor ions.
• A precursor ion is then subjected to the second round of fragmentation through collision and is broken into smaller ions known as product ions.

Analysis Strategies for Protein Identification

• In top-down strategy, intact proteins in a complex mixture are fractionated and separated into less complex protein mixtures or single proteins.
• The bottom-up strategy is commonly used for high-throughput analysis of small peptides derived from highly complex samples.
• Sort-then-break approach: Proteins in a complex mixture are first isolated and then enzymatically cleaved.
• Break-then-sort approach: Also known as the shotgun approach, wherein proteins are cleaved first and the resulting peptide mixture is then separated by LC and analyzed by tandem MS.

11.7: Microbial DNA Analysis for Bodily Fluid Identification

• Human Microbiota: Microbial community.
• Metagenomics: Allows the study of the microbial genome of complete microbial communities harvested directly from natural environments.
• The bacterial rRNA operon contains three rRNA genes:   * 5S and 23S rRNA genes encode the RNA components of the large subunit of the ribosome.   * 16S rRNA gene encodes the RNA component of the small subunit of the ribosome.
• The intergenic spacer region (ISR) between the 16S rRNA and 23S rRNA genes in the rRNA operon is another commonly used marker.

11.8: Nondestructive Assays for the Identification of Bodily Fluids

• Fluorescence: The emission of light by a fluorophore.
• Fluorophore: A moiety in a molecule that fluoresces on absorbing the energy from an excitation light source or radiation.
• Raman spectroscopy: Utilizes a near-infrared excitation light source and measures the scattering of laser light caused by the vibrating molecules of a sample.

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