LECTURE 2: SEROLOGY & BPA // CHAPTER 10

Question 1

Presumptive Tests

Answer 1

• 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

Question 2

Cellular Composition of Blood:

Answer 2

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

Question 3

Components of blood:

Answer 3

-	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

Question 4

Structure of Haemoglobin:

Answer 4

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

Question 5

Presumptive tests for blood:

Answer 5

-	Colour tests
o	Kastle-Meyer (Phenolphthalein)
o	Leucomalachite Green
-	Light-producing tests
o	Luminol
o	Crime-lites
-	Immunological

Question 6

Colour Tests

Answer 6

• Kastle-Meyer (KM) test (Phenolphthalein) = deep pink colour
• Leucomalachite green (LMG) = blue/green colour
• Reduced forms of KM and LMG are used = Colourless

Question 7

Use of Kastle-Meyer & Leuchomalchite Green

Answer 7

• 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

Question 8

Use of luminol

Answer 8

• 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

Question 9

Limitations of Luminol

Answer 9

• 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

Question 10

Other Tests For Blood:

Answer 10

• 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

Question 11

Presumptive Test for Saliva

Answer 11

• 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

Question 12

Saliva uses

Answer 12

• To identify a body fluid if there is a positive DNA result
• Sexual offences
• Murder
• Rape
• Spitting?

Question 13

Composition of semen

Answer 13

• 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

Question 14

Tests for semen

Answer 14

-	Presumptive: 
	Acid phosphatase (AP) testing
-	(more) Specific:
	p30 test for prostate specific antigen (PSA)
-	Confirmatory test:
	Microscopy

Question 15

Acid phosphatase testing

Answer 15

• 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

Question 16

P30 (Prostate Specific Antigen or PSA)

Answer 16

• 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

Question 17

Confirmatory Tests for Spermatozoa

Answer 17

• 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

Question 18

serology

Answer 18

• the study of bodily fluids left at crime scenes

Question 19

Presumptive

Answer 19

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

Question 20

Confirmatory test

Answer 20

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

Question 21

the major body fluids

Answer 21

blood, saliva, semen, urine

Question 22

Genetic markers in blood:

Answer 22

• 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

Question 23

BPA

Answer 23

• 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”

Question 24

1) Passive bloodstains:

Answer 24

include clots, drops, flows, and pooling

Question 25

2) Transfer bloodstains:

Answer 25

include wipes, swipes, pattern transfers, and general contact bloodstains

Question 26

3) Projected/ impact bloodstains:

Answer 26

include splatters, splashes, cast-off stains, arterial spurts or gushes

Question 27

Wipe stain

Answer 27

Created when an object moves through an existing bloodstain | e.g. wiping a rug on blood pool on floor

Question 28

Swipe stain

Answer 28

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

Question 29

Spatter stain

Answer 29

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

Question 30

Cast of stain

Answer 30

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

Question 31

Arterial spurts/ gushes

Answer 31

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

Question 32

Fly spot stains

Answer 32

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

Question 33

Voids

Answer 33

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

Question 34

‘skeletonized’ stain

Answer 34

- As a stain dries, the edges and borders dry first due to surface effects, if the bloodstain is removed

Question 35

Important measurements in BPA:

Answer 35

 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

Question 36

listing of bodily fluids that are subject to presumptive testing

Answer 36

``` blood saliva semen urine sweat mucus vaginal Fluid vomit faecal material ```

Question 37

define the qualities necessary for a test to be useful as a presumptive test

Answer 37

- rapid - safe - cheap - simple to perform

Question 38

discuss the importance of sensitivity and specificity

Answer 38

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.

Question 39

Confirmatory test for blood

Answer 39

Takayama test - results in the formation of salmon coloured crystals (heated in the presence of pyridine and a reducing sugar)