Serology


 * 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

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

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



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

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
 * Anitgen-Antibody Response** - when the immune system (white blood cells) recognizes a substance as foreign it attacks it in two ways

- 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



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



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)







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 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**
 * Blood Spatter**

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

Blood spatter video:

media type="youtube" key="Od8YuwUT794?fs=1" height="385" width="480"



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

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

www.prenhall.com/hsforensics

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?
 * __Forensic Characterization of Bloodstains__**

1. IS IT BLOOD? - 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
 * Presumptive Tests**

__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



- 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

- 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
 * Sangur sticks/Hemastix**





Catalytic Detection of non-visible Blood Stains - 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
 * Luminol**



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



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