The nature of blood

What is blood made of?
- mixture of cells, enzymes, proteins and inorganic substances

Plasma - the fluid portion of blood is called
- mainly water; composes 55% of blood make-up

The other 45% composes the solid part of the make-up
- red blood cells, white blood cells, and platelets are suspended in the plasma
- white blood cells contain DNA so can tell with certainty whether blood left at crime matches blood of a suspect or victim

Platelets - aid in blood clotting and repair of damaged blood vessels

Red Blood Cells (erythrocytes) - transport oxygen and help transfer carbon dioxide
- uses hemoglobin, an iron containing protein that binds oxygen
- responsible for the red color

White Blood Cells (leukocytes) - responsible for defense and immunity
- some surround, engulf and digest foreign invaders
- others secrete antibodies
- only blood cell that contains a nucleus so the only source of DNA



blood_distribution_pic.jpg












serum – the liquid that separates from the blood when a clot is formed


Antigens and Antibodies
antigen ¬- A protein that stimulates the production of antibodies
antibody - proteins secreted by white blood cells that attach to antigens

Anitgen-Antibody Response - when the immune system (white blood cells) recognizes a substance as foreign it attacks it in two ways
1. B lymphocyte (type of white blood cell) makes specific antibodies against the protein and bind the foreign substance
2. Phagocytes (type of white blood cell) engulf the invader


- there are millions of antigens on the surface of each red blood cell

- Blood antigens have been grouped into more than 15 systems depending on their relationship with one another, but the ones we will look at are A-B-O and Rh

Type O - 43%
Type A - 42%
Type B - 12%
Type AB - 3%

Type A individuals have A antigens on the surface
Type B individuals have B antigens on the surface
Type AB individuals have A and B antigens on the surface
Type O individuals have no antigens on the surface

cell_surface_proteins.jpg



Rh factor (aka D antigen)
Rh positive – people having the D antigen
Rh negative – people lacking the D antigen

Rh_factor_pic.jpg


The serum of the blood contains the proteins known as antibodies ( a protein in the blood that destroys or inactivates a specific antigen)
- the antibody is bivalent – it has two sites to attach to the antigen so it creates a cross-linked network that clumps the blood together (called agglutination)


general_structure_of_antibody.jpg



antibody_bonding_along_protein.jpg



agglutination.jpg



If anti-B (the antibody against the B antigen) is added to type B blood, the blood agglutinates.

Nature made it so that:

Type A blood contains anti-B but not anti-A
Type B blood contains anti-A but not anti-B
Type AB contains neither anti-A nor anti-B
Type O contains both anti-A and anti-B

See if you understand the material by playing the following game: http://nobelprize.org/educational_games/medicine/landsteiner/

Serology – the study of antigen-antibody interactions





Blood Spatter
- blood is a cohesive mixture - the molecules in the blood stick to each other
- sometimes blood can overcome these cohesive interactions and form secondary droplets called satellites

Read the Blood and Spatter Chapter pdf pages 10-13 and examine the link below

Blood spatter video:









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http://www.nfstc.org/links/animations/images/blood%20spatters.swf

http://www.bloodspatter.com/BPATutorial.htm





www.prenhall.com/hsforensics





Forensic Characterization of Bloodstains
Three questions to answer:
1. Is it blood?
2. From what species did the blood originate?
3. If the blood is human, how closely can it be associated with a particular individual?

1. IS IT BLOOD?
Presumptive Tests
- suggests but does not confirm the presence of blood; indicates that other tests should be performed
- negative result means the substance IS NOT blood
- positive result means the substance IS LIKELY blood

Types of Tests
1. Catalytic Tests

- tests based on the fact that heme from hemoblogin can catalyze breakdown of hydrogen peroxide
hemoglobin - Fe containing molecule in RBC that binds oxygen and carries it from the lungs to tissues
Blood contains hemoglobin (carries oxygen and makes blood red) which contains a heme group which contains an Fe which can catalyze the breakdown of hydrogen peroxide
- sensitivity: 1:100,000


heme.jpg



- usually produces a color change
- specificity: some vegetable materials such as potatoes and horseradish will also turn the test pink - PROBLEM WHEN OUTDOORS
- also, some metals interfere (Cu and Fe) - PROBLEM IF TESTING A VEHICLE
- if test is negative and blood is NOT present; if positive then might be blood

Catalytic Detection of Visible Blood Stains
Kastle-Meyer Test aka the phenolphthalein/peroxidase test

- when a blood stain, hydrogen peroxide and phenolphthalein reagent are mixed, the oxidation of the hemoglobin in the blood produces a deep pink color
- sensitivity: 1:10,000 (if 1 drop of blood were present in a bucket with 10,000 drops of water, the PHTH test would still turn pink)

- Testing Method: swab the stain with a q-tip or some filter paper, add a drop of PHTH, add of drop of peroxide
Kastle_Meyer_Reagent.jpg



Sangur sticks/Hemastix
- used in the field kit
- suggest if dried sample is blood
- positive result = green or yellow to blue-green
- Plastic strip with filter paper on tip containing TMB and peroxidase


hemastix_1.jpg


hemastix_2.jpg








Catalytic Detection of non-visible Blood Stains
Luminol
- also a presumptive test
- reaction between blood and luminol produces light instead of color
- sensitivity: most sensitive presumptive test - detects bloodstains diluted up to 300,000 times
- luminol reagent is sprayed onto items suspected to be blood and the room must be darkened
- any blood stains present produce a faint blue glow
- advantage: can screen large areas quickly and does not interfere with subsequent DNA testing


external image luminol-intro.jpg external image luminol-crimescene.jpg

Reaction: Luminol is C8H7O3N3
- luminol powder is mixed with a liquid containing H2O2 and some other chemicals (H2O2 and luminol are the major players)
- the reactions needs a catalyst to accelerate the process - in this case the Fe from hemoglobin is the catalyst
- also an oxidation reaction
- during the reaction, luminol loses N ad H and gains O to produce 3-aminophthalate
- the reaction leaves the 3-aminophthalate in an energized state as the electrons in the O atoms are in higher energy orbitals
- as the electrons fall to a lower energy level the extra energy is released in the form of light

Chemiluminescent Reaction Scheme
Chemiluminescent Reaction Scheme



source:
http://www.howstuffworks.com/luminol.htm



2. Color Tests/Crystal Tests - confirmation test

- heme from the hemoglobin molecule forms a crystal when reacted with certain substances

Microcrystalline Tests
- 2 common tests: Teichmann Test and Takayama Test

- small amount of blood added to microscope slide
- chemical solution added
- slide heated to form hemoglobin crystals (a pink crystal complex)
- crystals can be viewed under a microscope

- positive results indicate blood
- sensitivity: 0.001 mL of blood/0.1 mg hemoglobin
- 20 year old blood stains have given positive results



IS IT HUMAN BLOOD???

Precipitin Test
- Used to determine if the blood is of human or animal origin
- Basis: animals injected with human blood form antibodies against the blood
- the antibodies can be recovered by bleeding the animal and isolating the blood serum
*the serum is really human antiserum
- the antibodies recovered from the animal blood interact with the antigens in the human blood and clump

Most common Precipitin Test is the Precipitin Ring test
- human blood is layered on top of the animal serum containing the human blood antibodies
- the interaction between the antibodies and the antigens forms a cloudy ring at the interface of the two liquids

http://diverge.hunter.cuny.edu/~weigang/Images/18-03_precipitin_1.jpg


IS THE BLOOD RELATED TO THE CRIME OR A PARTICULAR INDIVIDUAL'S BLOOD?
Gel Diffusion and DNA fingerprinting