LECTURE 2: SEROLOGY & BPA // CHAPTER 10 Flashcards
Presumptive Tests
- Tests are used to indicate the presence of blood, semen and saliva and other
- These tests should be:
Rapid
Safe – cant be carcinogenic/ mutagenic
Cheap
Simple to perform
Cellular Composition of Blood:
Blood is composed of red blood cells (~45%), white blood cells (<1%) and plasma (~55%)
Plasma comprises 92% water, 8% protein and trace amounts of serum albumin, hormones, electrolytes, CO2 , blood clotting factors
Components of blood:
- White blood cells: o Immune response o DNA - Red blood cells: o Transportation of oxygen o Haemoglobin (250 million molecules per cell) o No DNA - Platelets o Clotting o No DNA
Structure of Haemoglobin:
Haemoglobin is a tetrameric compound with 4 haem groups
- Iron in the haem group is in the reduced state (Fe2+)
- One haemoglobin molecule carries 4 molecules of oxygen
- Therefore, each cell can carry 1,000 million oxygen molecules
- Haem has a peroxidase activity – This is used as the basis for most presumptive tests
Presumptive tests for blood:
- Colour tests o Kastle-Meyer (Phenolphthalein) o Leucomalachite Green - Light-producing tests o Luminol o Crime-lites - Immunological
Colour Tests
- Kastle-Meyer (KM) test (Phenolphthalein) = deep pink colour
- Leucomalachite green (LMG) = blue/green colour
- Reduced forms of KM and LMG are used = Colourless
Use of Kastle-Meyer & Leuchomalchite Green
- Both tests work in the same way
- Filter paper is folded to produce a corner
- The filter paper is rubbed over the stain for a few seconds
- A drop of the KM or LMG solution is added to the paper
- After a few seconds a drop of H2O2 is added
- A positive reaction will occur in a few seconds
- Fe2+ + H2O2 → Fe3+ + OH· + OH−
- H2O2 reacts with the iron in haem
- Cleaved into 2 hydroxyl ions
- Hydroxyl ions seek H2 to form stable H2O
- H2 donated from KM / LMG thus becoming oxidised and in their coloured form
Use of luminol
- Used for large areas such as rooms at a crime scene or areas suspected to be cleaned
- Produces a bright light in the presence of blood
- Dried and old bloodstains produce greater luminescence than fresh blood
- The luminescence fades within minutes
Limitations of Luminol
- Short-lived reaction therefore difficult to record result
- Possibly detrimental to further analysis
- Health & safety issues
- Can’t find fingerprints in blood by this method
- Can only be used as circumstantial evidence – e.g. if no blood is expected in an area and a lot is found
Other Tests For Blood:
- Benzidine
One of the oldest tests and works like KM & LMG but it is highly carcinogenic - Haemastix – designed to test if blood in urine
3,3’,5,5’-tetramethylbenzidine (TMB)
Simple to use and works like KM & LMG - Light Sources
Not a presumptive test but used to detect body fluids
Polylight, Quasar, Crime-lite
Presumptive Test for Saliva
- The enzyme α-amylase is present at very high levels in saliva
- Amylase catalyses the conversion of starch to simple sugars (glucose)
- Responsible for the primary digestion of starch and lipase fat in food
Amylase Test: - Paper containing homogenously linked starch polymers bound to a water soluble blue dye
- Amylase will break down this starch complex, releasing the blue dye
- The paper is moistened and pressed over the stain
o If a blue colour remains the stain does not contain amylase
o If a clear colour is produced amylase is present
Phadebas Amylase Test
Saliva uses
- To identify a body fluid if there is a positive DNA result
- Sexual offences
- Murder
- Rape
- Spitting?
Composition of semen
- Male ejaculate, or semen, is a mixture of several different components:
o sperm and seminal fluid (or plasma) consisting of testicular fluid and accessory gland secretions.
o The average amount of semen ejaculated is between 2‐5 ml.
o only 10% of that volume being sperm.
o between 20‐150 million sperm per mL. - Sperm
Spermatozoa
o Produced in the testes
o An ejaculate from a normal, healthy male contains 200 -500 million spermatozoa in 1.5 – 5 mL volume
o Dependent on many factors - Seminal fluid
secretion from the seminal vesicles, prostate and bulbourethal gland
Contains organic and inorganic components, e.g. Amino acids
Fructose
Acid phosphatase
Prostate specific antigen - The presumptive tests for semen detect specific proteins present in the seminal fluid, contributed by the prostate gland
Tests for semen
- Presumptive: Acid phosphatase (AP) testing - (more) Specific: p30 test for prostate specific antigen (PSA) - Confirmatory test: Microscopy
Acid phosphatase testing
- Acid phosphatase is present in seminal fluid at high concentrations
- AP converts α-naphthyl phosphate to αnaphthol which in turn combines with orthodianisidine to form a purple coloured complex
- The rate of the generation of the purple is proportional to the quantity of enzyme present
- Also in vaginal secretions – needs special consideration
P30 (Prostate Specific Antigen or PSA)
- A protein, called p30, appears to be present in semen and no other body fluid
- p30 is a glycoprotein produced in the prostate
- Antibodies against p30 produce a characteristic result
- Such an immunological test appears to be specific to semen
• More discriminating than AP
Confirmatory Tests for Spermatozoa
- Presumptive tests do not prove the presence of semen
- Confirmation can be carried out by microscopy
- Spermatozoa are much smaller than epithelial cells
- The presence of a tail aids greatly in the identification of spermatozoa
- H&E stain
Sperm heads are stained a pink colour and tails a dark purple
Haemotoxylin stains the nucleus and acrosome purple
Eosin stains the cytoplasm pink
Christmas Tree stain:
- So called because of the bright colours
- Utilises nuclear fast red that differentially stains the DNA containing head bright crimson
- A counter-stain of picric acid – indigocarmine (PIC) stains the tails green-blue-grey
serology
- the study of bodily fluids left at crime scenes
Presumptive
o Highly sensitive to but not specific to a particular substance
o e.g. colour of sample may indicate blood but not human vs. no human // or a false positive result, may not be blood just something else reacted in test
o The idea is that some false positives are acceptable but no false negatives are obtained
o These are then test with number 2
Confirmatory test
o Tests positive for substance in question and only that substance
o Lacks sensitivity
o Few false negatives are acceptable as long as no false positives are obtained
the major body fluids
blood, saliva, semen, urine
Genetic markers in blood:
- A blood group is a class of antigens produced by allelic genes at one or more loci and are inherited independently of other genes
- Genetically controlled and Invariant – robust marker for inclusion or exclusion
- About 20 human blood groups are known to exist
- Several systems are known to group blood
1) ABO blood group
1900, Karl Landsteiner
A, B, O refers to antigen on surface
BPA
- Bevel & Gardner (2002); the “analysis and interpretation of the dispersion, shape characteristics, volume, pattern, number and relationship of bloodstains at a crime scene to reconstruct a process of events”
1) Passive bloodstains:
include clots, drops, flows, and pooling
2) Transfer bloodstains:
include wipes, swipes, pattern transfers, and general contact bloodstains
3) Projected/ impact bloodstains:
include splatters, splashes, cast-off stains, arterial spurts or gushes
Wipe stain
Created when an object moves through an existing bloodstain
e.g. wiping a rug on blood pool on floor
Swipe stain
Transfer of blood onto a target by a moving object that is itself bloodstained
e.g. blood soaked clothing being dragged across floor is a swipe not a wipe
Spatter stain
Describes a stain that results from blood hitting a target. Two types.
Forward spatter – when droplets are projected away from the item creating the impact. E.g. hammer
Back spatter – caused by droplets being projected toward the item
Cast of stain
Result of blood being flung or projected from a bloody object in motion or one that suddenly stops
They are linear and reflect the position of the person moving bloody object
e.g. being bashed with a bat
Arterial spurts/ gushes
Blood flowing through arteries is under high pressure
These stains can vary due to the pumping action and variable pressure of the blood as it exits the wound, producing a zig-zag up-and-down pattern
Fly spot stains
Stains resulting from fly activity – and may mimic other relevant BPA patterns
Flies may regurgitate and defecate when consuming blood and can be confusing and misleading
Voids
Indicate that some secondary object came between a blood spatter and the final target, leaving an outline ‘shadow’ on the final target – gives insight into thing that may have been moved or removed but were there at present time
‘skeletonized’ stain
- As a stain dries, the edges and borders dry first due to surface effects, if the bloodstain is removed
Important measurements in BPA:
angle of impact is the acute angle created by the intercept of the target with the droplets vector.
Direction angle is the angle between the long axis of the stain and is standard reference point (0 degrees vertical).
Directionality demonstrates the vector of a droplet when it hits the target, tail points in direction of travel.
Satellite droplets are small amount fog blood that detach from the parent stain and ‘splash’ onto a surface
listing of bodily fluids that are subject to presumptive testing
blood saliva semen urine sweat mucus vaginal Fluid vomit faecal material
define the qualities necessary for a test to be useful as a presumptive test
- rapid
- safe
- cheap
- simple to perform
discuss the importance of sensitivity and specificity
Lalkhen and McCluskey define sensitivity as the ratio of true positives to the sum of true positives plus false negatives. They define specificity as the ratio of true negatives to the sum of true negatives plus false positives.
Presumptive tests must generally be sensitive and confirmatory tests must generally be specific.
Confirmatory test for blood
Takayama test - results in the formation of salmon coloured crystals (heated in the presence of pyridine and a reducing sugar)
