FRSC 675 Forensic Serology and DNA Analysis Exam 1 Review
Blood
-45% cellular (RBC ABO system) (WBC HLA system) (platelets) -55% Liquid/Plasma (clotting factors) (SERUM)
LeukoCrystal Violet
Blood Enhancement Technique, catalyzes decomp of hydrogen peroxide, heme reacting, oxidation forms purple crystal, used with porous surfaces
Amido Black
Blood Enhancement Technique, stains protein portion of blood blue-black
Coomassie Blue
Blood Enhancement Technique, stains protein portion of blood to blue-purple
Hungarian Red
Blood Enhancement Technique,stains protein, works well on non-absorbing surfaces, enhanced by fluorescence under green light
Vaginal Fluid
o Lubricating fluid secreted into the vagina o No component is specific, nor present in such high concentrations compared to other body fluids so there is no presumptive test to determine the prescence of VF o Prescence is inferred by the type of evidence
Blood Fxn
o Most prominent evidence at crime scene (usually dried) o 5-6 quarts in adult human § 8% body weight o Provides oxygen and nutrients to cells, takes away CO2 and waste, communication system, immunity, injury healing Circulates through entire body 20-30s
Hemastix strips
presumptive test for blood, green, not great sensitivity
Polyclonal Antibodies
produced in response to injection of a specific antigen, antigen may have many epitopes and hence can trigger many B-cells to produce antibodies against each epitope
ABAcard HemaTrace Test
Confirmatory for blood § Add sample at S site: conjugated mobile monoclonal antihuman Hb antibodies (MC Antibody) + (Dye) + (Hb Antigen if present in sample) § T site: immobile poluclonal antihuman Hb antibodies (MC Antibody) + (Dye) + (Hb Antigen) + (PC Antibody) gives a band § C site: immobile anti-immunoglobulin antibodies, bind excess MC antibodies from S site § Minimum detection level: .05 ug/mL blood § High dose hook effect: get 1 band at C site, too much blood so antigen migrates faster than MC antibody and dye so no band at T even though it is positive for human blood § Can get false pos with cat blood
Takayama Test
Confirmatory for blood,Heme + Pyridine + NaOH + Glucose inc. (75C) produce Pyridine ferroprotoporphyrin (pink feathery crystal)
Confirmatory Tests Semen
Microscopic ID of Sperm, Immunochromatographic Assay
Confirmatory Tests for Blood usage
Not commonly used: § Unnecessary sample consumption, not as sensitive as presumptive, more time consuming and costly, presumptive tests provide suitable reliability (few false positives especially with P-TMB)
Benzidine
Presumptive Test blood, carcinogenic, blue color (not used)
O-tolidine
Presumptive Test blood, carcinogenic, blue color (not used)
Leucomalachite Green (LMG):
Presumptive test for blood, o green, interferes with DNA typing, not great sensitivity
Rapid stain identification (RSID) Test
Saliva, § First rapid test for human saliva § Antibody target: human salivary alpha-amylase § No high dose hook effect (not much alpha amylase in saliva) § Sensitivity: 1:10000 § Cross reacts with rat saliva but no other animal § Confirmatory § False positive: some urine, breast milk, and feces
Monoclonal Antibodies
a collection of identical antibodies that interact with a single antigen site (epitope), derived from a single cell line (B-cell)
Luminol
blue chemiluminescence (enhancement test blood) § Luminol in ground state oxidized by Hb into excited state, blue chemiluminescence happens when electron returns to ground state § Reacts: · most strongly with aged bloodstains (Hb and RBCs broken down and heme more exposed) · equally with animal blood and human blood · with some metals (Cu), bleach, oxidizers, horseradish, salt-treated wood · nothing! (overspray causes auto-luminescence)
Antiserum:
collection of antibodies o Purified blood serum containing entire spectrum of antibodies produced by host animal o Inject host with proteins from species that you want to develop antiserum for à allow time for immune system of host animal to build antibodies à belled host animal and separate the serum (no clotting but all proteins) à purify and test specificity of antiserum (has antibodies against specific antigen)
Collection of Blood, Purple-topped
contains EDTA anticoagulant, liquid is plasma (serum+clotting factors), DNA analysis (most common)
Collection of Blood, Gray-topped
contains NaF, for alcohol/toxicology/drug testing
Advantages Luminol
extremely sensitive (1:300,000 dilution detectable), doesn't interfere with DNA typing but too much dilution impact Takayama Test
Fluorescein/HemaScein
green fluorescence (blood enhancement test) Fluorescin (Stays in red state in prescence of Zn), oxidized by Hb to fluorescein, fluoresces under alternate light source
Probative
item of evidence that moves towards proving the charge
Collection of Blood, Red-topped tube
no preservatives, will clot, liquid is serum
Disadvantages Luminol
not specific to blood (Cu, horseradish, and bleach also react), chemiluminescence only lasts ~30s, need dark conditions, maybe carcinogenic
Forensic Examination of Bloodstains (4 types of tests)
o Blood enhancement (not visible to naked eye): luminol, not specific o Presumptive tests: quick and easy, low LOD, can give false positives o Confirmatory tests: more time consuming, done in lab, higher detection limit, highly specific, no false positives o Species identification: immunological, DNA
Urine components
o Components: § Water, soluble wastes, excess sugar, urea, inorganic ions, uric acids, creatinine, citric and lactic acids, epithelial cells, WBCs (infection), bacterial cells (lactobacillus)
Urine detection
o Detection Use ALS to locate, difficult to obtain DNA from urine (centrifuge large volumes to pellet WBCs (need ~100mL)
Forensic Serology
o Detection, identification, and typing of body fluids and secretions, either in native form or as stains or residues left at a crime scene o Used exclusively from 1950s-1980s but not used as screening tool prior to DNA analysis o Essential first step of evidence analysis o Time consuming and labor intensive
Menstrual blood ID
o Discrimination of menstrual blood vs. venous blood o Importance: rape, missing female (evidence of injury or normal cycle?) o Target: Matrix Metalloproteinases (MMPs) § Protein highly expressed in menstruating endometrium, MMP7 and 11 (11 more specific but MMP7 more sensitive and preferred) o qPCR to amplify MMP7 mRNA, use Ct (lower cycle # Ct more protein to begin with)
Urine
o Fluid excreted by kidneys containing metabolic waste from blood o Average volume 1L/day depending on fluid intake ([] of waste dependent on fluid intake) o Types of evidence: Dried stains or liquid samples (need a lot to get DNA)
Composition of semen
o Liquid: seminal fluid § Lubrication for motility of sperm cells § Secreted by urinogenital glands § Contains many proteins, salts, and organic molecules § Provides nourishment to sperm o Solid: cellular material § Sperm (spermatozoa) · Single cell: spermatozoa or spermatozoan · Male reproductive cell · Produced by testes, mature/stored in epididymis § Epithelial cells · Sloughed from linings of vas deferens, ejaculatory duct, bladder, and urethra
o Central dogma molecular biology
§ DNA transcribed to mRNA translated to protein § Reverse transcription mRNA to DNA § Replication DNA to DNA
Combined P-TMB Test
o Phenolphthalein or Kastle-Meyer test: Pink (P-TMB test) o Tetramethylbenzidine: blue (P-TMB Test) § Combines sensitivity and specificity utilizing two presumptive tests for blood § P-test requires basic environment, TMB requires acidic § P-Test more specific than TMB § TMB slightly more sensitive than P-Test § Positive result must come from both tests
Urine presumptive tests
o Presumptive tests § Urease test (most common) · Urea + urease = ammonia + carbon dioxide (changes pH) · Liver breaks down amino acids producing urea for excretion, urine has high amount compared to other body fluids · Also present in sweat, blood, seminal fluid, saliva, vaginal fluid · Not confirmatory § Creatinine Test · Creatinine + picric acid = orange creatinine picrate crystals · Not popular b/c picric acid toxic and carcinogenic § Sulfate Test · Inorganic sulfates + barium chloride = insoluble white precipitate § Chloroplatinic Acid Test · Urea + chloroplatinic acid = yellow bi-pyramidal crystals
Advantages of Serology over DNA
o Provides information on source of the DNA o Fast and cheap way to screen samples (DNA testing ~2 days) o Helps in paternity cases
Saliva secretion
o Secreted in mouth by salivary glands § Parotid (largest), submandibular (deep in floor of mouth), and sublingual (smallest pair, front of mouth just under tongue), minor salivary glands found throughout the mouth
Semen and Seminal Fluid production
o Semen produced in testis and mature in epididymis then pass through vas deferens and collects liquids from three glands: seminal vesicle, prostate gland, and Cowper's gland (bulbourethral gland) then goes out the urethra o Single ejaculation: 2.5 to 6 mL of seminal fluid, 200-500million sperm
Composition of Seminal Fluid
o Seminal vesicles 60% § Choline (chemiluminescence) § Spermine § Spermidine (unique component of semen, not 100% unique but higher concentrations compared to other fluids) o Prostate gland ~30% § Acid phosphatase (AP test) § P30/Prostate specific antigen (PSA, confirmatory test) (unique component of semen) o Epididymis and Bulbourethral gland ~10% § Mucous/lubricant o Miscellaneous § Water, urea, citric acid, fructose, calcium, potassium, magnesium, zinc, flavins(fluorescence)
§ MicroRNA basics
o Small non-coding RNA molecules o 18-25 nucleotides o Partially complementary to one or more mRNA o Silence protein synthesis, Downregulate gene expression (translational repression, mRNA cleavage, de-adenylation) · Advantages: more stable than mRNA, also present in DNA extracts (less sample consumption), stable with environmental contaminents · Disadvantages: suitable target mRNA are not available for all body fluid, requires standardization by housekeeping gene
Cellular material (semen)
o Sperm cell (55um) § Head: 4x2x1um, ovoid, acrosome 1/3 of head can be dif color on slide, flat base § Tail and mid-piece: length ~50um, not always visible on slide o Epithelial Cell (9-17um) § Squamous, large cytoplasm, small nucleous
Semen evidence
o Stains: bed sheets, underpants, clothing o Sexual assault evidence kits: vaginal, anal, oral, and body swabs Condoms
Saliva components
o Thin colorless digestive fluid (1-1.5 L/day) o Components § Liquid · 97-99% water · Mucin: lubricates, protects teeth, inactivates bacteria · Proteins: histidine rich proteins (buffering), IgA/IgM (immune function) · Salts · Enzymes: Peroxidase (less than in blood), amylase (very common, most tests), maltase (digestive), lysozyme (kill bacteria), alkaline phosphatase § Cellular · Buccal epithelial cells, leukocytes, bacteria (strep, all saliva), yeast
Antibody Structure
o Two light and two heavy polypeptide chains, held together by disulfide bridges o Light chain has constant and variable, antigen binding regions o Heavy chain has constant and variable antigen binding regions o 2 active binding sites (variable regions)
Other biological fluids
o Typically not screened, extracted directly for DNA or examined microscopically by a pathologist for confirmation o Bone, hairs, tissue, perspiration/skin cells (use ALS, fingerprint chemicals), cerebrospinal fluid (CSF), amniotic fluid, bile, meconium, vomit, breastmilk
· DSI-Semen
o Uses DNA-methylation, confirmation of sperm DNA o Automatable, high throughput, sensitive o HhaI (RE) methylation, cleaves unmethylated GCGC o Looked for loci that are consistently methylated in specific tissues or unmethylated in specific tissues o Specificity: differentiated semen from saliva, venous blood, menstrual blood, urin, vaginal fluid (100%) o Sensitivity: more sensitive than traditional sperm slide searches, 1 of 33 items tested positive for sperm DNA when no sperm found on slide
Forensic examination of semen stains (5 types)
o Visual exam § Dried stains white/yellowish, crusty/stiff, diffuse stains on dark surfaces may appear powdery, not specifc o Enhancement § Flavins fluoresce when exposed to 450nm ALS, blood can quench fluorescence, not specific o Presumptive § Unique components of seminal fluid · AP (most common), choline, spermine, spermidine · Quick and easy, low detection limits, crime scene and lab, false postives o Confirmatory § More time consuming, lab, higher detection limit, highly specific § P30/PSA, sperm under microscope (100% confirmatory, need large sample) o Species ID § Immunological p30/PSA § Microscopic § DNA
Chain of custody
o a written record accounting for the whereabouts of evidence at all times
Presumptive Tests for blood Basic principle:
o based on peroxidase properties of Hb, Heme cleaves oxygen free radicals from peroxide group and these free radicals oxidize organic chemicals (dyes), presumptive test detects oxidized organic dyes
· mRNA basics
o exists as usable mobile copy of DNA to make proteins o only exists in cell if those particular proteins are made in cell (tissue specific) o some proteins only found in certain body fluids thus their mRNA are only found in those fluidso Fragility of mRNA (less stable than DNA) § Samples sitting for years, degraded, fungus/bacteria/mold contamination, samples treated with heat, samples treated with bleach or other chemicals o Recent studies have shown mRNA stable for months even 2-15 years
Serum
o yellowish liquid that separates from clotted blood § Contains proteins and antibodies § Does not contain cells, clotting factors or fibrinogens
Indication
possibility that body fluid is there without using chemicals (alternate light source), presumptive
Starch Iodine Test
saliva, § Digest sample + starch solution + iodine at 37 C get free iodine and yellow color if positive § If negaticve no change from original blue color § Disadvantage: subject to interference by proteins, blood, and iodine-sequestering lipids
Phadebas test
saliva, § Phadebas: insoluble starch cross-linked with blue color dye · Crush up and put in water and add sample, digest at 37 C (body temp b/c enzymatic reaction) · Clear in upper water area neg, blue in upper water area pos (bottom always blue bc powder sinks to bottom, liquid tells result) · Advantage: not affected by prescence of other proteins · Disadvantage: not a confirmatory test · False Positives: feces (pancreatic amylase), breask milk, some VF, plant materials
SALIgAE test
saliva, § Proprietary mechanism based on prescence of alpha-amylase § Available as spray for location or in tubes for samples § Positive: clear to yellow § Negative: no color change § False postitives: expirated blood (from mouth), some urine, breast milk, and feces § Cross-reacts with many animal salivas § Sensitivity ranges from 1:10-1:2000 (less sensitive than RSID)
Procion Red Amylopectin (PRA) Paper Test
saliva,§ Paper treated with PRA(byproduct of starch production, glucose polymer) and incubated with sample at 37 C for 15 min · Positive: pink to white · Negative: pink color remains § 98% of clothing shows amylase + stains before washing
· Crossover electrophoresis
species ID o Antigen/antibodies driven to each other by electric field o Very similar to ODD, use electrophoresis to make gel run faster instead of natural diffusion o Electroendosmosis (EEO): flow of solvent in electric field during electrophoresis, anode (+) to cathode (-) o White band pos 3-5uL per well
Precipitation Reaction
species ID o Extensive cross-linking between Ag-Ab complexes occurs o Lattice structures that are formed begin to aggregate together forming a visible complex o Key reaction for all immunological methods
· Zone of equivalence
species ID o No unbound antigen or antibody (need similar amounts of each important) o Prozone: excess antibody, no agglutination o Postzone: excess antigen, not enough antibody to result in complete reaction
· Tube precipitin
species ID, o Antigen/antibodies layered onto each other o Double diffusion test using liquid-liquid interfaces o Antiserum in bottom of tube and layer sample on top, positive if a white line forms between layers (50uL each layer) o Incubate 10-15min
Chromatography (immunochromatographic)
species ID, o Antigens combine with dye-tagged mobile antibodies o Complex diffuses across areas (control and test lines) containing immobilized antibodies o ABAcard Hema trace, ABAcard p30, RSID saliva, Seratech, BLUESTAR Identi-PSA o High dose hook effect ***
Ouchterlony Double Diffusion (ODD)
species ID, time consuming, o Both antigen and antibody diffuse to zone of equivalence o Agarose gel (1%) § Punch holes in gel, place antiserum in center hole and samples including host pos and neg controls in surrounding holes § Proteins diffuse from origin into the agarose gel § If antiserum recognizes antigens in sample will form lattice à visible line § Pos: visible white line (location depends on amount antigen antibody, want right in the middle) , neg: no line § Takes a long time: incubate at room temp overnight or 37 C 3-4hrs
Specificity
test for specific fluid or species, false + or - possible?
Detection
use chemical test to prove there is significant evidence body fluid on evidence likely is ___ (colorimetric test), presumptive
Identification
use one or several tests that focus on presence of unique/innate property of a body fluid, no doubt based on tests that body fluid is [blank], confirmatory
Leukocytes (WBCs)
§ 1 WBC per 600-700 RBCs, many dif types, immune response, large
Erythrocytes (RBCs)
§ 40% blood volume, 4-6 million per uL, biconcave discs, no nucleus (mammals), high Hb content 97%, circulate 4 months then broken down into bile by liver
Hemoglobin
§ 97% of RBC dry content, gives red color (Fe content, unique to blood), MetalloProtein (complex tetramer. 68kD, four subunits each is globular protein with Heme group) · Fe atoms (4) allow for reversible binding of oxygen molecules, each Hb can bind 4 O2 § Only Fe2+ binds oxygen (not Fe3+)
o Item
§ A unit of evidence (can be single or collective) § Place initials lab #, and item # on outside and on unstained/unaffected area of item § Preserve the evidence (dry, document everything)
o Assaults/violent crimes against persons
§ Associate 2 people, victim and perpetrator § Was suspect/victim bleeding?
Thrombocytes (platelets)
§ Cell-like but no nucleus, smaller than RBCs and WBCs, 1 platelet per 20-30 RBCs, essential in clotting, produced in bone marrow
Secondary Biological Fluids (not many tests for these)
§ Cerebrospinal fluid § Amniotic fluid § Bile § Vomit § Breast milk
o Dual duty as scientist and consultant
§ Conduct DNA analysis and advise investigator about lab capabilities and advantages/disadvantages of analysis
o Technical Aspects of Species ID (QC)
§ Controls: host control (serum of host animal used to produce antiserum), positive control, negative/substrate control § Before using a lot number, specificity of antiserum must be tested and documented · All available sera should be tested against antiserum § Documentation · Diagram representation of placement of samples and observations, date of testing, initials of person doing testing § Storage of antisera · Aliquots are frozen within 1 week of reconsitituion · Thawed aliquots are good refrigerated for 1 month
o Evidence routing in multi-section cases
§ DNA before drugs (pipes, bags, smoking devices) § DNA and bloodspatter cooperate § DNA before latents (consult may be necessary) · Wear cotton gloves under latex, any obvious ridge detail don't swab over it § DNA before tox (DNA requires less sample) § DNA before documents exam (cotton gloves under latex) § DNA before firearms § Trace depends on exam · Fibers DNA first · Anthrax trace first § DNA typically first because it is more easily degraded by the other analyses (only need pen tip amount for analysis)
o "Used" Items
§ Describe fully § Envelopes: pry apart seal with forceps and scalpel; swab seal § Duct tape: freeze ~ 1 week and attempt to peel apart, cut ~1/2 in squares don't touch ripped edge § Bottles/cans: swab mouth area § Cigarette butts: cut ~1/8" of filer paper at mouth end § Ball caps: swab sweatband § Airbags: if no blood swab all over
Amylase
§ Digestive enzyme- breaks down carbohydrates (starch) · Starch is not water soluble unless broken down, amylase breaks it down so attaching a dye to a starch so that a color change occurs when it is broken down is a good strategy for detection § Produced in parotid gland § Found in high concentrations in saliva in primates and omnivores § Absent in carnivore and herbivore saliva § Requires pH 6.6-6.9 § AMY genes: · AMY 1 gene (salivary): expressed in saliva, perspiration, breastmilk · AMY 2 (pancreatic): expressed in pancreas, semen, vaginal secretions § Found in other body fluids but at >6000 times lower than saliva · Feces sometimes equivalent levels (pancreatic) § Human saliva has >1000 times higher amylase than animal saliva
o Note taking
§ Document everything thoroughly (most cases don't go to trial until 2-4 years after analysis) one line strikethrough and initial § Every page contains: date, FS Lab No, Original Initials § First Note: container · Outermost package of item · Initial, lab #, and item # on outside · Record: container # and description, sealed/unsealed, initialed/uninitialed
o Take custody
§ Documented on RFLE § Doc internal lab transfers on separate internal transfer form § Final transfer back to agency from lab documented on RFLE
SOPs
§ Everyone follows the same procedure § Consistency § Validated § Prevents/minimizes errors
RSID-Urine
§ First rapid test for human urine § Antibody target: 60kD Tamm-Horsfall Protein (THP; uromodulin) § Mild high dose hook effect § Sensitivity 10uL § Confirmatory but, THP present in all mammalian urine. Cross reacts with some animal urine (gorilla, dog, horse, rat) § False positive not reported with other body fluids § Inhibited by presence of blood
o Post examination
§ Go back to case file § Call investigator if any new questions or info come up § Reconcile notes with RFLE and correct if necessary § Prepare for DNA analysis
o Epigenetics
§ Heritable changes in gene expression (not due to changes in DNA sequence) § DNA sequence is same in different kinds of cells § Different tissues of human body cannot be differentiated by DNA sequence but can be differentiated by epigenetic patterning § Modification: DNA methylation change cell behavior but not DNA sequence
· Chromatography (immunochromatographic) Disadvantages
§ High dose hook effect § Dirty samples can result in false negative § No longer considered confirmatory for body fluid § False positives for other body fluids
Common hosts
§ Horse: gives sharp precipitin bands (antigen and antibody bind very tightly), requires narrower optimal precipitin zone § Rabbit: cost effective, more stable and diffused precipitin bands § Goat: preferred choice for large scale production because size
Blood Colors
§ Human and other vertabrates: red Hb (Fe) § Crustaceans: blue hemocyanin (Cu) § Segmented worms: green, chlorocruorin § Unsegmented worms: violet, Hemerythrin § Luminol reacts with Cu or Fe so can confuse red and blue blood
o Breaking and entering
§ ID perpetrator and connect with scene § Choose most probative (blood first, inside house rather than outside)
o Juvenile & adult rapes
§ If the victim knows what happened only examine samples associated with offense § If not(young child/mentally challenged/drugged/drunk rapes) examine everything
o New tech for BFID
§ Immunochromatographic (catalytic, immunologic) (Non-DNA)§ Sperm Identification Systems (help find sperm under microscope) (Non-DNA) § DNA methylation § Messenger RNA § MicroRNA -Raman Spectroscopy§ Microbial Signature
Species ID
§ Immunological in nature (use antibodies created to react in visible manner with a species' biological fluids) § Newer techniques are DNA or RNA based: PCR amplification kits, mRNA profiling, 12S ribosomal RNA, Mitochondrial DNA sequence analysis
o Requestion for Laboratory Examination (RFLE)
§ Includes crime data/type, victim and suspect names race DOBs, agency case #, statement of fact § Starts chain of custody (signed over to lab) Internal transfers go through separate lab documentation
Sperm Morphology
§ Intact: 0-36 hrs in vagina, 2-6 hrs in rectum and mouth § Head only: 7 days in vagina, 24-36hrs in rectum, 24hrs in mouth § Post coital changes · Some start ~16hrs o Darkening and shrinking of acrosome, formation of vacuoles in nucleus and acrosome, darkening of cell membrane around nucleus o Difficult to ID severely deformed head, keep looking for better one § Factors that lead to low or no sperm: alcoholism, diabetes, cancer, local infection, frequent ejaculation, vasectomy
o General notes on Examinations
§ Item, distinguishing markings, size, brand § Condition § Size, shape, color, and heaviness of stains § Photograph if necessary § Results of body fluid testing § Amount of sample taken for DNA and amount remaining § Fabric separations noted (tears) § Hairs/fibers noted § Clothing: fasteners type and number, fabric content, pocket contents § Swabs: number, color and amount staining, results of testing § Weapons: manufacturer, type, size, condition, parts, stains or lack thereof, tissue, hairs/fibers § Guns: describe appropriately, manufacturer and model #, serial number, parts, call in firearms examiner if clarification needed
QC
§ Set of activities for ensuring quality in the actual product § Day-to-day operational techniques and activities used to fulfill QA § Product oriented § Focus on identification and correction (implement correction in QA)
o PCR based Methods
§ Species ID via mitochondrial DNA sequencing of 12s rRNA gene · Same primers amplify 11 dif species · 20bp hypervariable region is different for each species § Advantages: no antiserum QC, sensitive § Disadvantages: expensive, complicated and time consuming
ODD Disadvantages
§ Takes time § Lose some concentration of samples due to double diffusion
Presumptive Tests semen
§ Kaye's/Walker/Acid Phosphatase test · Done in lab · AP found in very high levels in semen (high up to age 40 then decrease) but also in other body fluids (vaginal fluid, saliva, CSF, urine) differentiate by how quick the reaction is (high [] react within 10s) · Not related to sperm count, little variation with fertility/vasectomy · Advantages: very quick, doesn't require prior visualization, can detect uo to 500X diluted semen · Disadvantages: sensitivity decreases with age and storage, AP present in other body fluids · How it works: o AP from prostate gland reacts with alpha-Naphthyl Acid Phosphate and either fast blue or fast red to give a color within 10s if positive · False positives: fecal material, vaginal fluids, urine, saliva, plant material (cabbage, cauliflower, brussels sprouts), fungi (mushrooms and yeast), bacteria (vaginal infections) · AP mapping o Transfer semen sample from clothes to filter paper by pressing down 10-15 min then add substrate and dye to paper to do the test
Sensitivity
§ LOD
Hairs as evidence
§ Last resort unless specifically requested § Collect using post it note and examine under stereomicroscope § Look at root and for obvious tissue cut ½" root end with tissue § Need root for DNA
False Positives blood
§ Luminol and BlueStar: copper and nickel salts (in coins, plumbing, wiring) (color: copper green, nickel pink, pressure-treated wood green, blood red to black) (rate of color change slower with false positives) § All screening tests: oxidizing agents (bleach and water treatments, nitrites to cure meat, gunshot residue, other explosive residues containing nitrocellulose, nitroglycerine, nitrates, nitrites) § Screening Tests (acid environment): Plant peroxidases (vegetable material onion, garlic, beet, cucumber, bread gluten, horseradish) (note appearance of stain) (plant peroxidases temp sensitive)
For each submission
§ Make sure RFLE matches submission (descriptions and number containers, sealed condition, single line strikethrough) § FS Lab ID on packages and RFLE § Sign into custody
Utility of DNA analysis
§ Match suspect with evidence § Paternity testing (~19 min) § Missing person investigation § Mass disasters (match body parts) § Convicted felon DNA database § Species ID/wildlife poaching § Microbial ID (not quite to individualization but 92% accurate in IDs)
o Challenges with existing BFID methods
§ Methods are either presumptive or confirmatory but destructive § Require separate tests for each body fluid § Marker need additional steps (RNA-based methods, more sample consumption and more labor intensive, convert to DNA) § Markers not stable over time mRNA based methods § Multiplexing of multiple primer sets expensive (time consuming)
Confirmatory Tests for Blood Types
§ Microcrystalline tests: view under microscope, not robust, difficult to interpret · Teichmann and Takayama § Antibody tests (immunochromatographic) · ABAcard and HemaTrace
Uritrace Test
§ Modified creatinine test § Chromatographic method for urine detection § Commercialized Picric acid or creatinine testing · Creatinine combines with picric acid to form orange creatinine picrate (15min)
· Crossover electrophoresis Disadvantages
§ More complex apparatus (but common for molec bio)
ODD Advantages
§ Multiple samples can be tested at once § Requires less reagents than tube precipitin § Works even for dirty samples (gel acts as filter) § Optimum Ag to Ab concentration allows for precipitation § Sensitive compared to other methods
· Chromatography (immunochromatographic) Advantages
§ No antiserum/sera QC § Simple, inexpensive § Little sample prep § Sensitive up to 1:100 blood § Easy to perform and read results
Presumptive tests for feces
§ Organoleptic examination: sight and smell § Microscopic examination: view for plant and goblet cells, starch granuels, bacteria, yeast, muscle fibers, and parasites, hairs may be visible in animal feces § Chemical tests: Edelman's test for urobilinogen § Edelman's test · Bilirubin (blue pigment) reduced by bacteria to urobilinogens (intestine) oxidized to urobilins that are present in the fecal sample (brown color) add zinc chloride solution which creates Urobillin-Zn chelate then add either UV or white light and see crab apple green (UV) or rose pink (white) fluorescence (may be orange in herbivores due to chlorophyll) · Incubate at 55 C
55% liquid/plasma (clotting, serum)
§ Plasma (91% water, 7% protein, 2% electrolytes): liquid portion, separated after addition of anticoagulants § Serum: liquid portion of blood w/o clotting factors, separated after blood allowed to clot § Buffy Coat: white interface between plasma and RBCs following centrifugation, contains WBCs
· Tube precipitin Advantages
§ Quick and reliable, little prep time
Primary biological fluids(after blood & semen)
§ Saliva § Urine § Feces § Perspiration § Vaginal fluid § Menstrual fluid
Enhancement and visual exam (semen)
§ Semen fluoresce under alternate light or UV b/c of flavin and choline-conjugated proteins (blue UV, yellow ALS) § Advantages: quick, good for field § Disadvantages: not good for all samples (affected by heat, humidity, microorganisms), many other molecules fluoresce in similar way § Not all semen stains will fluoresce (substrate, concentration) § Not all fluorescent stains are semen (false positives: sweat, saliva, nasal mucus) § General rule of detectability: · Better: flat fabrics, concentrated, dark/plain fabric (underpants, t-shirt, toilet paper) · Worse: soft/fuzzy fabrics, diluted stain, light/patterned fabric (sweatpants, bath towels)
o What is needed in new BFID method?
§ Sensitive, specific, confirmatory § Does not consume all evidence sample § Does not add additional step (method that can utilize same DNA for BF and individualization) § Can identify majority of BF in single test § Can handle mixture § Can handle old cases where only DNA available § Can be easily implemented in next-generation sequencing forensic panel or existing set-up of forensic labs § Is resistant to environmental degradation § With known error rate if any
QA
§ Set of activities for ensuring quality in processes by which products are developed (follow SOP exactly) § Planned and systematic actions § Process oriented § Focus on defect prevention
Forensic analysis of saliva
§ Use ALS to locate (screening) § DNA techniques more sensitive than the screening test § Common tests are all amylase detectors § No amylase detected does not mean saliva is not there (saliva is mostly water so diluting it further can make it very hard to detect, more difficult than blood)
ABO blood system
§ Used for blood typing, antigen/antibody reaction · If blood cells A, anti-B antibody in serum · B, anti-A antibody in serum · AB, no antibodies · O, both antibodies · Rh antigen, + · No Rh, - § No longer used for forensic analysis § Used before DNA available § Still useful for: bone marrow and organ transplants and blood transfusions
Homicides
§ Usually have pre-submission conferences § 50-100 submissions (Item #s important) § Communication with investigators key
· Crossover electrophoresis Advantages
§ Very sensitive 10-8g of protein § Inexpensive (better than tubes, might need more antiserum than ODD) § Fast compared to ODD § Multiple samples processed at once
· Tube precipitin Disadvantages
§ Vulnerable to contamination (no dirty extracts, no gel to filter) § Expensive (uses lots of antiserum) § One sample per tube
o Things to ask at submission
§ What exams requested? § Any wet items or need for refrigeration? · Maggots? Bloody and wet? PERKS have blood tubes and possible oral rinses in them. Tox evidence (urine, blood, tissue) § Any undried biological evidence? § Guns/knives appropriated secured, unloaded and packaged? § Any fragile evidence?
Antiserum specificity
§ depends on components used to sensitize host animal · Univalent: specific to one antigen · Polyvalent: containing several antibodies each capable of reacting with specific antigen § Antisera made in closely related animal will result in greater specificity § Human antisera also reacts with baboon, higher primates
Advantages Fluorescein/HemaScein
§ extremely sensitive (1:100,000 dilution), doesn't interfere with DNA typing, fluorescence lasts longer than luminol (4-5min), can be used in lighted condition
Two types of immune system
§ innate or non-specific (macrophages) and adaptive or acquired (lymphocytes such as B-cells (produce antibodies) and T-cells)
Disadvantages Fluorescein/HemaScein
§ not specific to blood (Fe/Cu can cause fluorescence), external ALS required, maybe carcinogenic
Feces
· (not a fluid) o Excretion product of digestive system o Importance: anal sodomy, inanimate object penetration, sent in the mail o Composition: § Water 75% § Solids 25% · Bacteria 30% (gut bacteria, 50% weight of fecal material) ·Fiber/components of digestive juices (30%) · Fat 10-20% · Inorganic matter 10-20% Protein 2-3%
§ Immunochromatographic (catalytic, immunologic) (Non-DNA)
· ABAcard HemaTrace, previously covered (Blood) o Confirmatory test for blood, human specific, 10min after transferred to plate · Bluestar OBTI (Blood) o Antibody: human Hb, Dye: red/pink o Reported studies: § High dose hook effect at neat and 1:10 blood, sensitivity 1:2,000,000 0.05ug/mL Hb § Does not claim to be confirmatory, cross react with primates o VCU studies: § Sensitivity 1:7500 § Neat blood no HDH § Can cross react with primates § No false pos with other body fluids § Sig better results when using proprietary buffer than water · Bluestar Identi-HEM (Blood) o Antibody: Human Hb, Dye: colloidal gold o Manufacturer reports: § Sensitivity 0.01ug/mL Hb § No cross reactivity with other body fluids or animals § Does not claim to be confirmatory · RSID-Blood o Antibody: glycophorin A (RBC membrane protein), Dye: colloidal gold o Manufacturer validation: § No HDH, sensitivity 50-100nL of blood, no cross reactivity with other body fluids or animals *best* · PSA Semiquant (semen) o Antibody: Human PSA, Dye: colloidal gold o Manufacturer reports: § Sensitivity 2ng/mL, internal control 4ng/mL, no false pos/neg, no HDH o Crime lab validation: § Outperformed ABAcard p30, sensitivity 167 pg/mL, no false positives o 3 bands so 2 controls control and internal standard · RSID-Semen o Antibody: human semenogelin (seminal vesicle), Dye: colloidal gold o Manufacturer validation: sensitivity 1uL, no cross reactivity with other BF or animals, high dose hook effect occurs ~50uL neat semen o Studies report: sensitivity 1:100,000, false positive with male urine, HDH occurs
Microscopic ID of Sperm
· Doesn't work if infertile or vasectomy · Wet mount o Immerse cutting of stain in water directly on slide, differentiate human sperm from other species o Field bright, cant see as well, better with phase contrast microscope · Christmas tree stain (K-PICS) o Kernechtrot solution: acrosome colorless, heads color o Picroindigocarmine solution: neck blue, tails green o Stain takes a long time, need a lot of material · Other stains: o Hemotoxylin-Eosin Staining: acrosome pink, head blue, tail pink o Papanicolaou stain: H-E stain + 3 stains for epithelial cells, mainly for cervical cancer screening · Bright field vs. Phase contrast o Unstained § Bright field may be difficult to see § Phase contrast heads glow, acrosome appears dark o Stained § Not very different in visibility o Generally phase contrast inc visibility · Sperm HY-Liter o All nuclei stained with DAPI fluorescent dye (stain DNA) o Sperm stained with fluorescently-labeled antibody specifc for sperm head protein o FITC fluorescence filter microscopy filters all but the fluorescent tag
§ Raman Spectroscopy
· Each body fluid yields unique spectrum · Advantage: Non-destructive · Disadvantages: not validated on several body fluid samples, costly to integrate into DNA crime lab workflow, variations between individuals, mixed body fluid samples, contaminated samples, interference from various substrates, venous versus menstrual blood
§ Microbial Signature
· Humans have more bacterial cells than human cells · More bacteria than human in feces and urine · Method for BFID: o Sample prep à extract DNA à qPCR analysis à library prep (1 primer pair universal bacterial primer but sequence different for dif bacteria, single primer to ID all body fluid) à high throughput sequencing à data analysis · Bacterial signature differ in different body fluids · Disadvantage: bacterial structure associated with menstrual and vaginal fluids not significantly different · Bacterial signature associated with human body fluid can be utilized for accurate body fluid ID 83-100%
§ Sperm Identification Systems (help find sperm under microscope) (Non-DNA)
· LAI: uses KPICS staining, automated scanning up to 3 slides, computer memory holds photos of potential sperm, keeps XY coordinates of each candidate · NicheVision: KPICS or H&E staining, automated scanning up to 4 slides, specializes in low sperm #, distinction between sperm and WBC or heavy epithelial load, fully automated sperm ID, 106.28% +/- 115.37% more spermatozoa than with manual exam · Computer memory holds photos and XY coordinates
§ DNA methylation
· Methyl groups added to C5 position of cytosine of DNA · Nearly 80% of CpGs are methylated in mammals (5-C-Phosphate-G-3) · Important in embryonic and normal development · Regulate gene expression pattern · For forensic use to help differentiate tissue type · Easy integration into forensic workflow o DNA extraction à DNA digestion by RE (cleaves unmethylated restriction site) àmethylation specific PCR o DNA extraction à bisulfite conversion (convert unmethylated cytosine to uracil) àmethylation specific PCR or HTS § Different base pairs have different melting curve b/c double vs triple bond § Can be detected by high resolution melt curve analysis, SNaPshot, HTS § Advantages: Less sample consumption (same DNA can be used for both individualization and BFID), stable § Disadvantages: larger inter-personal variations, variation with age and environmental factors, not good for mixture analysis, need large amount DNA
Positive Ctrl
· Obtain specific known body fluid and test to make sure test working properly
§ Immunological Methods (antigen-antibody) species ID
· Precipitation Reaction · Zone of equivalence -Ouchterlony Double Diffusion · Tube precipitin · Crossover electrophoresis · Chromatography (immunochromatographic)
Antibodies
· Substance specific tag that host gives to foreing substance (antigen) o Provide an immune response to antigen, directly neutralizes antigen, tags antigen for removal/destruction by other components
Combined P-TMB Test Procedure
· Swab small area of stain, place small cutting on filter paper, add 1 drop water, add 1 drop EtOH to cleanse (remove dirt, fats/oils, expose heme, no reaction yet), add 1 drop phenolphthalein (pink color change false positive, hint of green may appear believed to be KOH breaking down RBCs), add 1 drop H2O2 (immediate pink color change positive reaction), add 1 drop TMB (phenolphthalein reverts to colorless due to acidic environment, immediate green-blue color change of TMB indicates positive reaction)
How to use luminol
· Turn of lights wat 5 min for eyes to equilibrate then spray fine mist using long broad strokes then apply in well-ventilated room then bottle 1 ft away from surface then do not allow surface to saturate or streak
How to use Fluorescein/HemaScein
· Turn off lights wait 5 min, spray fine mist, spray 3% hydrogen peroxide, view using ALS and orange filter
mRNA BFID
· Used for blood and saliva ID · Menstrual blood ID· seminal fluid and vaginal fluid ID
Immunochromatographic Assay (semen)
· Works if infertile or vasectomy · P30 card o Antigen-antibody reaction § Antigen: PSA/p30 (prostate) § Antibody: human specific, also found in very low levels in brest milk and amniotic fluid (false positives very uncommon) o Works like ABAcard S, T and C sites o High levels PSA in semen 0.2-3mg/mL · No longer considered confirmatory for seminal fluid o Indication still very strong · Don't need as much sample and quicker than microscopic exam · High dose hook effect: o Occurs when p30 concentration too high o Unbound antigen moves faster than antigen-antibody dye complex
Immunoglobulin (Ig) molecules
· proteins) o An Ig molecule with a specific amino acid sequence o Gives each Ab the ability to adhere to and interact with only one antigen
Negative Ctrls
· reagent blank: only test reagents no substrate · substrate control: substrate and reagents o make sure substrate does not cause false positive · water: water and reagents, may include substrate
§ MicroRNA BFID method
· small RNA isolation à barcoding and library prep à parallel deep sequencing of all samples in one flow cell à separate sample sequences based on unique identifier tag àindentify all miRs in each sample and normalize à calculate relative abundance of each miR, identify miRs diagnostic for each body fluid, identify potential normalization miRs · Similar to mRNA, not as specific
