Fundamentals of Serology & Microscopy PDF

FUNDAMENTALS OF SEROLOGY & MICROSCOPY BIO 440 SEROLOGY  Purpose  identify cell types on an item and/or  identify the type of stain present  May help determine the best route for DNA analysis  How much DNA is expected to be present  Male...

FUNDAMENTALS OF SEROLOGY & MICROSCOPY BIO 440 SEROLOGY  Purpose  identify cell types on an item and/or  identify the type of stain present  May help determine the best route for DNA analysis  How much DNA is expected to be present  Male/female component  Method to use  TWO CLASSIFICATIONS OF TESTS  Presumptive  Aka preliminary tests  Indicates that the stain may be of a certain origin  Does not conclusively prove what it is  Used to screen large items and to determine what test to do next  Good: narrows down the possibilities  Bad: high risk of false positives, too sensitive  TWO CLASSIFICATIONS OF TESTS  Confirmatory  Conclusively determines the origin of the stain  May be one or multiple procedures  Good: less risk of false positives  Bad: more expensive, more equipment, longer procedures  FALSE RESULTS  False positive: A positive result when in fact the result should be negative  False negative: a negative result when in fact the result should be positive  Example: pregnancy tests FLUIDS TESTED IN SEROLOGY  Body fluids commonly tested that we will be covering:  Blood  Semen  Saliva  Other body fluids that can be tested:  Urine  Feces  Menstrual fluid GENERAL CLASSIFICATIONS OF TESTS  Visual examinations  Naked eye, alternative light  Colorimetric assays  Color-changing  Chemiluminescent assays  Generates light  Immunochromatographic assays  Uses antibodies BLOOD BLOOD  Serology test results reported as:  Positive presumptive test: the evidence may be blood  Confirmatory test: the evidence does contain blood  Blood constitutes 8% of body weight  Blood consists of  Plasma  Red blood cells (RBC) or erythrocytes  White blood cells or leucocytes  Platelets BLOOD  Plasma  Fluid in which the blood cells live  90% water, 10% proteins, minerals, hormones, antibodies  Red blood cells  Life span of 3 months  Contain no nucleus  Contains hemoglobin  Protein that brings O2 to cells, brings CO2 to lungs  Can also bind CO and CN, cause chemical asphyxia HEMOGLOBIN Heme molecule in a subunit of hemoglobin Kobilinsky & Levine (Eds.). (2011). Forensic chemistry handbook. Retrieved from http://ebookcentral.proquest.com.lopes.idm.oclc.org/lib/gcu/reader.action? docID=697683&ppg=293 BLOOD  White blood cells  Defend the body against infection  Contain a nucleus  Responsible for the DNA source in blood  Platelets  Aid in blood clotting  Collect in areas of vascular injury  Contain no nucleus WORKFLOW FOR BLOOD TESTING Kobilinsky & Levine (Eds.). (2011). Forensic chemistry handbook. Retrieved from http://ebookcentral.proquest.com.lopes.idm.oclc.org/lib/gcu/reader.action? docID=697683&ppg=293 IDENTIFICATION OF BLOOD: VISUAL EXAMINATION  Appears candy-apple red or deep red  Flaky and cracks if dried on a smooth surface IDENTIFICATION OF BLOOD: VISUAL EXAMINATION  Appears black under ALS  Cannot say it is blood without testing  “Visually consistent with blood” IDENTIFICATION OF BLOOD: COLORIMETRIC ASSAY  Phenolphthalin Assay, aka Kastle-Meyer Test  Oxidation-reduction reaction catalyzed by heme  Oxidation is loss of hydrogen  Reduction is gain of hydrogen  Phenolhthalin is the reductant  Hydrogen peroxide is added as the oxidant  When heme is present, a colorless reductant is oxidized and produces a color  Presence of pink color indicates a positive result IDENTIFICATION OF BLOOD: COLORIMETRIC ASSAY  Phenolphthalin Assay, aka Kastle-Meyer Test  The colorless reductant phenolphthalin is oxidized to phenolphthalein by heme and peroxide, producing a pink color Kobilinsky & Levine (Eds.). (2011). Forensic chemistry handbook. Retrieved from http://ebookcentral.proquest.com.lopes.idm.oclc.org/lib/gcu/reader.action? docID=697683&ppg=293 IDENTIFICATION OF BLOOD: COLORIMETRIC ASSAY  Heme is the catalyst for the reaction  In the presence of heme the solution will turn pink  Pink result gives a presumptive positive for the presence of blood IDENTIFICATION OF BLOOD: COLORIMETRIC ASSAY  Very sensitive  Detects down to 10-5 fold dilution  Not specific for blood  False positives (heme not present)  Oxidants  Household cleaners  Bleach  Plant peroxidases  Horseraddish  False negatives  Reductants  Inhibit oxidation reaction IDENTIFICATION OF BLOOD: COLORIMETRIC ASSAY  How is it done?  Transfersmall amount of stain onto moist swab OR cut out very small sample (1 mm2)  Add one drop of alcohol  Add one drop of KM reagent  If it turns pink here, false positive—start over  Add one drop of peroxide  Positive—turns pink within 1 minute  Negative—turns pink after 1 minute IDENTIFICATION OF BLOOD: CHEMILUMINESCENCE ASSAY  Light is emitted as a product of the chemical reaction when viewed with UV light  Givesa presumptive positive result for the presence of blood  Can be sprayed over large suspected bloodstained areas  Detects small blood droplets and blood traces  Detects patterns and prints IDENTIFICATION OF BLOOD: CHEMILUMINESCENCE ASSAY  Luminol is oxidized by heme to produce a chemiluminescent light Kobilinsky & Levine (Eds.). (2011). Forensic chemistry handbook. Retrieved from http://ebookcentral.proquest.com.lopes.idm.oclc.org/lib/gcu/reader.action? docID=697683&ppg=293 IDENTIFICATION OF BLOOD: CHEMILUMINESCENCE ASSAY  Good:  Shows cleaned areas of scenes  Detects down to 10-3 fold dilution  Bad:  Dilutes blood, leading to lower DNA levels  Room must be completely dark to see the light  Same false positives/negatives as KM IDENTIFICATION OF BLOOD: CHEMILUMINESCENCE ASSAY  Blue Star  Good:  Does not require 100% darkness  Does not require UV light  Does not interfere with obtaining DNA profiles  Bad:  Same false positives/negatives as KM  Does dilute DNA IDENTIFICATION OF BLOOD: IMMUNOCHROMATOGRAPHIC ASSAY  Confirmatory test for both blood AND species (human)  Uses antibodies to detect human antigens  Antigens injected into animal  Animal produces antibodies  Blood drawn, antibodies removed and purified  Polyclonal antibodies: contains mixture of antibodies against various human antigens  Monoclonal antibodies: specific, react with only one site on the antigen  No cross-reactivity with commonly encountered animals, but do with higher primates IDENTIFICATION OF BLOOD: IMMUNOCHROMATOGRAPHIC ASSAY  Detects human glycophorin A (GPA)  Protein present on the surfaces of human red blood cells  What kind of protein is this? IDENTIFICATION OF BLOOD: IMMUNOCHROMATOGRAPHIC ASSAY  How to do it:  Take small cutting of the sample  Place in microcentrifuge tube  Add universal buffer  Sit for 10 min to 2 hours  Add liquid to sample window  Read results at 10 minutes IDENTIFICATION OF BLOOD: IMMUNOCHROMATOGRAPHIC ASSAY  How it works:  Sample is added to the sample window  Mobilized anti-GPA antibody binds to GPA, creating a complex  In the test zone the GPA in the complex binds to the immobilized antibody  A pink line is created at the test line  Unbound anti-GPA binds to immobilized antibodies at the control line  A pink line at the control line IDENTIFICATION OF BLOOD: IMMUNOCHROMATOGRAPHIC ASSAY  Can read results BEFORE the 10 minute mark, but not after  Can be difficult to see positive results for dilutes samples IDENTIFICATION OF BLOOD: IMMUNOCHROMATOGRAPHIC ASSAY  Reading the results  Control line indicates test worked properly  This proves the test was valid  Test line indicates the presence of human blood IDENTIFICATION OF BLOOD: IMMUNOCHROMATOGRAPHIC ASSAY  Good:  No cross-reactivity with blood from other organisms or primates  Sensitive for low levels of blood (down to 10 -11 (100 nL))  Bad:  Testline can be very faint  Reaction areas don’t always line up with letters  Can get false negatives  Done incorrectly  Too dilute SEMEN SEMEN  Normal semen contains seminal fluid and sperm cells  Typical ejaculation releases 2-5 mL of semen that contains 107-108 spermatozoa/ mL  Spermatozoa are produced in the testes, sent to mature in the epididymus to mature for 3 months  SEMEN  Combines with seminal fluid in the prostatic urethra  60% of ejaculate comes from the seminal vesicles  Containsugar, amino acids, citric acid and prostaglandin hormones  30% of ejaculate fluid comes from the prostate  Contains acid phosphatase (AP) and prostate-specific antigen (PSA)  Contains citric acid, minerals and enzymes SEMEN  5% comes from the epididymis  Contains the electrolytes potassium and sodium and some nutrients for the sperm  5% comes from the bulbourethral gland  Secretes a small amount of mucus for lubrication  Flavin causes semen to fluoresce in UV light SEMEN  Vasectomy is the surgical removal of a portion of the vas deferens  Prevents sperm from reaching the reproductive tract  May cause aspermia—males who produce no sperm  Oligospermia—men with an abnormally low sperm count  Aspermic and oligospermic males still produce ejaculate IDENTIFICATION OF SEMEN: VISUAL EXAMINATION  ALS/FLS can be used to search for possible semen stains  Flavins & choline-conjugated proteins fluoresce  Wavelength of 450-495 nm with colored goggles allows for visualization  Appears blue or yellow, depending on wavelength  Shows moderate to high fluorescence  Moderate for older stains IDENTIFICATION OF SEMEN: VISUAL EXAMINATION  Not specific for semen  Vaginal fluid  Urine  Saliva  Mayonnaise SEMEN: ACID PHOSPHATASE (AP)  The prostate is the most abundant source of AP in the body  Also found in lysosomes and other cell types, body tissues  This makes AP testing presumptive  Vasectomies do not affect AP levels  Half-life is 6 months at 37°C  Decreased if stored in wet environment  Extremely high AP levels in the blood can indicate cancer IDENTIFICATION OF SEMEN: COLORIMETRIC ASSAY  AP will hydrolyze the phosphate ester α-naphthyl phosphate  Acidphosphatase is the enzyme  α-naphthyl phosphate is the substrate  Phosphate group is removed via hydrolysis  The presence of brentamine Fast B solution forms a purple precipitate IDENTIFICATION OF SEMEN: COLORIMETRIC ASSAY  How to do it:  Transfer stain to swab or filter paper, OR take a small cutting (1 mm2)  AP is water soluble  Place one drop of AP spot solution onto swab or stain  Positive if purple color develops within 1 minute  After 1 minute results are not reliable IDENTIFICATION OF SEMEN: COLORIMETRIC ASSAY  Can use it to map a large item  Separate item into different areas  Moisten large piece of filter paper  Press different paper onto each area  Test with AP spot solution  Purple areas are traced back to their origin on the garment SEMEN: PROSTATE-SPECIFIC ANTIGEN (PSA OR P30)  Major protein present in ejaculate  Produced in prostate  Also found in sweat, perianal, & mammary glands & blood (prostate cancer)  Can be detected in sweat, feces, urine, & breast milk, blood  Half-life in a dried stain is 3 years at room temperature  Decreased if stored in wet conditions IDENTIFICATION OF SEMEN: IMMUNOCHROMATOGRAPHIC ASSAY  P30 testing cassettes can be used  Works exactly the same  Sample is added to the sample window  Anti-P30 antibody binds to P30, creating a complex  In the test zone the P30 in the complex binds to the immobilized antibody  A pink line is created at the test line  Unbound anti-P30 binds to immobilized antibodies at the control line, creating a pink line IDENTIFICATION OF SEMEN: IMMUNOCHROMATOGRAPHIC ASSAY IDENTIFICATION OF SEMEN: IMMUNOCHROMATOGRAPHIC ASSAY  High-dose hook effect  Caused by a very high concentration of antigens  Unbound antigen reaches the test line before the complex containing the antigen does  The colloidal gold-labeled antibody on the antigen creates the pink line  Results in:  decreased intensity at test line  false negative  Manufacturer claims this will not be encountered under normal working conditions  Could this be a problem? SEMEN: SPERMATOZOA  Consists of head, middle piece, and tail SEMEN: SPERMATOZOA  Head contains nucleus and acrosomal cap  Acrosomal cap enzymes break down outside of egg for fertilization  Middle piece contains mitochondria to power the tail  Tail is responsible for mobility  Contains flagellum  https://www.youtube.com/watch?v=65BV5dXXxzM SEMEN: SPERMATOZOA  60% of sperm have normal morphology IDENTIFICATION OF SEMEN: MICROSCOPY  Why is this a confirmatory test?  Cells are transferred to a microscope slide using water  Cells are heat-fixed with low heat  Apply Christmas tree stain to slide  Red dye first—stains all nuclei  Green dye second—stains all cell membranes IDENTIFICATION OF SEMEN: MICROSCOPY  How to do it:  Cells are transferred to a microscope slide using water  Cells are heat-fixed with low heat  Apply Christmas tree stain to slide  Red dye first—stains all nuclei & sperm cell acrosomal caps  Green dye second—stains sperm neck & tail, all cell membranes  Add cover slip  Assess under microscope  Cells can be removed from the slide for DNA analysis if necessary IDENTIFICATION OF SEMEN: MICROSCOPY Epithelial Cell Epithelial Cell Nucleus Debris IDENTIFICATION OF SEMEN: MICROSCOPY  Sperm Hy-Liter  Immuno fluorescence staining kit  Specific – identifying human sperm heads only  Sensitive – helping find hidden sperm  Flexible – allowing to identify sperm on hospital smear slides or from extracts in just seconds  Differential staining allows for the identification of different cell types  Sperm cells  Epithelial cells SALIVA SALIVA  1.0 to 1.5 L of saliva produced daily  70% from the submandibular salivary glands  25% from the parotids  5% from sublingual salivary glands SALIVA  Consists of mostly water, small amounts of electrolytes, antibodies, buffers, glycoproteins, and enzymes  Amylase enzyme begins to break down food in the mouth  Cleaves polysaccharides with α linkages  Cleaves off one maltose (2 glucose) at a time SALIVA  β-amylase found in plants & bacteria  Human α-amylase have two isomers  Pancreatic α-amylase, made by the pancreas (HPA)  Salivary α-amylase, made by the salivary glands (HSA) SALIVA  Amylase activity also found in  Breast milk  Blood  Tears  Semen  Vaginal secretions  Sweat  Urine  Stable for a few weeks to a few months IDENTIFICATION OF SALIVA: VISUAL EXAMINATION  ALS can be used to search for possible saliva stains  Not specific for saliva  Wavelength of 470-555 nm with colored goggles allows for visualization  Shows light fluorescence  May only see the edges of the stain IDENTIFICATION OF SALIVA: IMMUNOCHROMATOGRAPHIC ASSAY  Human α-amylase testing cassettes are used  Presumptive or confirmatory?  Manufacturer: confirmatory because of the minimum concentration needed to obtain a positive result  Industry: positive results have been obtained from human body fluids (especially feces) and α-amylase from other mammals  So which is it? IDENTIFICATION OF SALIVA: IMMUNOCHROMATOGRAPHIC ASSAY  Same procedures as for previous cassettes  Sample is added to the sample window  Anti-amylase antibody binds to α-amylase, creating a complex  In the test zone the α-amylase in the complex binds to the immobilized antibody  A pink line is created at the test line  Unbound anti-amylase binds to immobilized antibodies at the control line, creating a pink line STANDARD OPERATING PROCEDURES (SOP)  Outlines how the testing will be performed  Provides the steps, NOT the purpose  Do we need to know the purpose?  Each method or procedure has its own SOP  Method must be properly validated before use  Demonstrates that the method works at that lab  Improper validation causes stop of work until proper validation and approval  All employees MUST follow the same procedure STANDARD OPERATING PROCEDURES (SOP)  Reviewed during lab audits  Deviations from the SOP MUST be recorded in the notes along with reasoning  Deviations MUST be approved by the lab supervisor prior to use  Unapproved deviations can cause results to be invalid  What does that mean for court? For the evidence?  Not properly following SOP can make you appear to be incompetent and incapable  Can ruin your career  You are unreliable  You cannot be trusted