Forensic SerologyForensic Serology

Chapter 12Chapter 12

Karl LandsteinerKarl Landsteiner

In 1901 he discovered that all humans do not In 1901 he discovered that all humans do not have the same blood typehave the same blood type

He discovered the A-B-O blood group systemHe discovered the A-B-O blood group system

This saved millions of lives by preventing This saved millions of lives by preventing mismatched blood transfusionsmismatched blood transfusions

Other researchers were able to discover the Rh Other researchers were able to discover the Rh factor because of his researchfactor because of his research

29 years later, they gave Karl a Nobel Prize 29 years later, they gave Karl a Nobel Prize

Other Blood FactorsOther Blood Factors

The ABO proteins and Rh factor The ABO proteins and Rh factor proteins are not the only proteins on proteins are not the only proteins on the surface of blood cellsthe surface of blood cells

There are more than a 100 different There are more than a 100 different surface blood factors surface blood factors

But the ABO factors are most But the ABO factors are most important for matching a donor to a important for matching a donor to a recipientrecipient

Blood and ForensicsBlood and Forensics

No 2 individuals (except identical twins) No 2 individuals (except identical twins) can share all the 100+ blood factors can share all the 100+ blood factors

So forensic scientists used this tool to So forensic scientists used this tool to link blood from a crime scene to an link blood from a crime scene to an individualindividual

After 1990 however, DNA fingerprinting After 1990 however, DNA fingerprinting has been used instead of blood typing has been used instead of blood typing to identify blood stains and other to identify blood stains and other biological evidence.biological evidence.

What is Blood?What is Blood?Various cell types found in a

liquid matrix called plasma

• Erythrocytes (RBCs)– Transport O2, CO2

– Stay within blood vessels

• Leukocytes (WBCs)– Fight infection (immune

system)– Can move from blood

vessels into tissues

• Platelets (Cell fragments)– Help with clotting

Platelets

White Blood Cell

Red Blood Cells

Characteristics of Red Blood Characteristics of Red Blood CellsCells

Red blood cells (erythrocytes) are biconcave disks Red blood cells (erythrocytes) are biconcave disks that contain oxygen-carrying hemoglobin.that contain oxygen-carrying hemoglobin.

Red blood cells discard their nuclei, mitochondria Red blood cells discard their nuclei, mitochondria and most organelles during development and so and most organelles during development and so cannot reproduce or produce proteins.cannot reproduce or produce proteins.

In addition to 100s of cell surface proteins, their cell In addition to 100s of cell surface proteins, their cell surfaces have a special class of blood factor surfaces have a special class of blood factor proteins called proteins called Antigens.Antigens.

There are at least 15 different antigens, but the There are at least 15 different antigens, but the most important are the most important are the ABO and Rh antigensABO and Rh antigens

Role of RBCsRole of RBCs Transport inhaled OTransport inhaled O22 from lungs to all body from lungs to all body

cellscells

Remove CORemove CO22 from all body cells and take it from all body cells and take it back to lungs for exhalationback to lungs for exhalation

When oxygen combines with hemoglobin When oxygen combines with hemoglobin bright red oxyhemoglobin results.bright red oxyhemoglobin results.

Deoxygenated blood or blood containing CODeoxygenated blood or blood containing CO22 (deoxyhemoglbin) is darker.(deoxyhemoglbin) is darker.

RBCs are destroyed after about 120 days RBCs are destroyed after about 120 days

Summary of Functions of WBCs Summary of Functions of WBCs

Leukocytes can squeeze between cells lining walls of blood vessels by diapedesis and attack bacteria and debris.

Neutrophils and monocytes are phagocytic, with monocytes engulfing the larger particles.

Eosinophils moderate allergic reactions as well as defend against parasitic infections.

Basophils migrate to damaged tissues and release histamine to promote inflammation and heparin to inhibit blood clotting.

Lymphocytes are the major players in specific immune reactions and some produce antibodies (T cells and B cells)

Blood Platelets Blood Platelets

Blood platelets are fragments of cellsBlood platelets are fragments of cells

Platelets help repair damaged blood vessels Platelets help repair damaged blood vessels by adhering to their broken edges.by adhering to their broken edges.

Thrombopenia, or low platelet counts Thrombopenia, or low platelet counts increase bleeding risks increase bleeding risks

Thrombocytosis or high platelet count may Thrombocytosis or high platelet count may lead to thrombosis (clotting, bruising, strokes, lead to thrombosis (clotting, bruising, strokes, heart attacks). heart attacks).

Blood Plasma

• Plasma is the clear, straw-colored fluid portion of the blood.

• Plasma is mostly water, with a mixture of, amino acids, proteins, carbohydrates, lipids, vitamins, hormones, electrolytes, and cellular wastes

HematocritHematocrit

A blood hematocrit is normally 45% cells and 55% plasma.A blood hematocrit is normally 45% cells and 55% plasma.

Hematocrit means the percentage of red blood cells in blood

Buffy Coat?Buffy Coat?

Buffy coat is the layer that contains most of Buffy coat is the layer that contains most of the white blood cells and platelets. the white blood cells and platelets.

““Buffy” because it is buff (color of naked skin) Buffy” because it is buff (color of naked skin) in colorin color

The buffy coat is used to extract DNA from The buffy coat is used to extract DNA from blood - RBCs have no DNA, only WBCs do.blood - RBCs have no DNA, only WBCs do.

ABO Blood GroupABO Blood Group

Type A blood has A antigens on red blood cells and Type A blood has A antigens on red blood cells and anti-B antibodies in the plasma.anti-B antibodies in the plasma.

Type B blood has B antigens on red blood cells and Type B blood has B antigens on red blood cells and anti-A antibodies in the plasma.anti-A antibodies in the plasma.

Type AB blood has both A and B antigens, but no Type AB blood has both A and B antigens, but no antibodies in the plasma.antibodies in the plasma.

Type O blood has neither antigen, but both types of Type O blood has neither antigen, but both types of antibodies in the plasma.antibodies in the plasma.

Adverse transfusion reactions are avoided by Adverse transfusion reactions are avoided by preventing the mixing of blood that contains matching preventing the mixing of blood that contains matching antigens and antibodies.antigens and antibodies.

– Adverse reactions are due to the agglutination of red blood Adverse reactions are due to the agglutination of red blood cells.cells.

Rh Blood GroupRh Blood Group The Rh factor was named after the rhesus The Rh factor was named after the rhesus

monkey.monkey.

If the Rh factor surface protein is present If the Rh factor surface protein is present on red blood cells, the blood is Rh positive; on red blood cells, the blood is Rh positive; otherwise it is Rh negative.otherwise it is Rh negative.

There are no corresponding antibodies in There are no corresponding antibodies in the plasma unless a person with Rh-the plasma unless a person with Rh-negative blood is transfused with Rh-negative blood is transfused with Rh-positive blood; the person will only positive blood; the person will only then then developdevelop antibodies for the Rh factor. antibodies for the Rh factor.

Blood Groups and Transfusions Blood Groups and Transfusions Antigens and AntibodiesAntigens and Antibodies

Clumping of red blood cells following Clumping of red blood cells following transfusion is called agglutination.transfusion is called agglutination.

Agglutination is due to the interaction Agglutination is due to the interaction of proteins on the surfaces of red blood of proteins on the surfaces of red blood cells (antigens) with certain antibodies cells (antigens) with certain antibodies carried in the plasma.carried in the plasma.

Only a few of the antigens on red blood Only a few of the antigens on red blood cells produce transfusion reactions.cells produce transfusion reactions.

– These include the ABO group and Rh group.These include the ABO group and Rh group.

Agglutination of RBCs

Antibody: a protein that destroys or inactivates a specific protein antigen. Antibodies are found in the blood serum

Antibodies are very SPECIFIC to an antigen. They are designed by the immune system to fit an antigen.

Antiserum: blood serum in which there are specific antibodies

Agglutination: the clumping together of red blood cells by the action of an antibody

Precipitin: an antibody that reacts with its corresponding antigen to form a precipitate

  Secretor: an individual who secretes his or her blood-

type antigen(s) in body fluids. Approximately 80 percent of the population are secretors

Enzyme: a type of protein that acts as a catalyst for certain specific reactions

Iso-enzymes: multiple molecular forms of an enzyme, each having the same or very similar enzyme activities

Antibodies or Agglutinins Proteins that are present in the serum responsible for ensuring that the only blood

cells that can survive in a person are cells of the correct blood type

Type O Blood Possessed by people whose genotype is OO

both parents passed on the O gene have no antigens

these cells can be introduced into a person with Type A or Type B because these cells are not attacked by the antibodies these people possess

have both a & b antibodies can only have other O type cells mixed with this

blood

Relative Frequency of Blood Types in Human Populations

Population O A B AB US whites .453 .413 .099 .035 US blacks .491 .265 .201 .043 Chinese .439 .270 .233 .058 Eskimos .472 .452 .059 .017 Armenians .298 .499 .132 .080 Bolivian Indians

.931 .053 .016 .001

DRUG - DETECTION

Hypothesis: Since foreign substances in blood are identified

and then isolated by the immune system, it should be able to detect the presence of drugs and other chemicals.

This hypothesis is incorrect. The immune system only creates antibodies and launches attacks against foreign proteins (either free proteins or ones bound to cells) and not against other chemical compounds.

We can fool the immune We can fool the immune systemsystem

The drug can be attached to a carrier The drug can be attached to a carrier molecule that is a proteinmolecule that is a protein

This drug-protein combo can be injected This drug-protein combo can be injected into an animal such as a rabbit or rat (no into an animal such as a rabbit or rat (no human volunteers)human volunteers)

The animal’s immune system will create The animal’s immune system will create antibodies that are specific to the shape of antibodies that are specific to the shape of this drug-protein combo moleculethis drug-protein combo molecule

We can isolate these antibodies from the We can isolate these antibodies from the animal’s blood serum animal’s blood serum

And use these antibodies to detect the And use these antibodies to detect the presence of the drug in human blood or presence of the drug in human blood or urineurine

Polyclonal AntibodiesPolyclonal Antibodies

Usually when the immune system Usually when the immune system produces antibodies, they are a produces antibodies, they are a mixture of immunoglobulin molecules mixture of immunoglobulin molecules designed to recognize and bind to designed to recognize and bind to many different sites on an antigen many different sites on an antigen molecule or different molecule or different epitopesepitopes. In . In other words, they are made against a other words, they are made against a specific antigen, but each recognizing specific antigen, but each recognizing a a different epitope different epitope on the antigen. on the antigen. These antibodies are called These antibodies are called Polyclonal antibodiesPolyclonal antibodies

Polyclonal antibodies are not Polyclonal antibodies are not very specific or reliablevery specific or reliable

Because the animal makes so many Because the animal makes so many different kinds for ONE antigen, the different kinds for ONE antigen, the antibodies may not be consistent and antibodies may not be consistent and some antibody molecules may not some antibody molecules may not work very well.work very well.

Monoclonal AntibodiesMonoclonal Antibodies

Monoclonal antibodies are those that Monoclonal antibodies are those that are not only specific to one antigen, are not only specific to one antigen, but specific an exact epitope on that but specific an exact epitope on that antigen.antigen.

In other words, it is a collection of In other words, it is a collection of identical antibodies that bind to a identical antibodies that bind to a single antigen site or epitope.single antigen site or epitope.

How are polyclonal antibodies made?How are polyclonal antibodies made?

1.1. Inject the animal with the protein Inject the animal with the protein (antigen) and wait for a period of time for (antigen) and wait for a period of time for the animal to make polyclonal antibodiesthe animal to make polyclonal antibodies

2.2. Then collect the animal’s serum which Then collect the animal’s serum which will contain the polyclonal antibodies.will contain the polyclonal antibodies.

How are monoclonal antibodies made?How are monoclonal antibodies made?

1.1. Inject the animal with the protein (antigen)Inject the animal with the protein (antigen)2.2. Remove the spleen and isolate spleen cells Remove the spleen and isolate spleen cells

(the spleen produces the antibodies)(the spleen produces the antibodies)3.3. Fuse the antibody-producing spleen cells with Fuse the antibody-producing spleen cells with

tumor cells that were already growing in tumor cells that were already growing in culture (these are now called hybridoma cells)culture (these are now called hybridoma cells)

4.4. Grow the hybridoma cells in culture and isolate Grow the hybridoma cells in culture and isolate the cells that are producing the antibody of the cells that are producing the antibody of your choice.your choice.

5.5. These cells will be making antibodies that bind These cells will be making antibodies that bind to only ONE epitope on the antigen. The cells to only ONE epitope on the antigen. The cells can be maintained indefinitely.can be maintained indefinitely.

6.6. The antibodies are therefore called monoclonal The antibodies are therefore called monoclonal antibodies.antibodies.

Now how does a criminalist use these monoclonal antibodies?

To detect drug presence in blood, urine, etc. A.K.A. EMIT assays (bio jargon for test) EMIT stands for Enzyme-multiplied

Immunoassay Technique EMIT is most often used to test for marijuana

(THC) metabolites in urine. One of the primary THC metabolites is THC-

9-carboxylic acid

How EMIT worksHow EMIT worksLet’s suppose you’re checking someone’s urine for the Let’s suppose you’re checking someone’s urine for the

presence of THC-9-carboxylic acid (from THC or presence of THC-9-carboxylic acid (from THC or Marijuana.)Marijuana.)

1.1. Take urine sample to be testedTake urine sample to be tested2.2. Add monoclonal antibodies for THC-9-carboxylic acid Add monoclonal antibodies for THC-9-carboxylic acid

to the urineto the urine3.3. The antibodies will immediately bind to any THC-9-The antibodies will immediately bind to any THC-9-

carboxylic acid molecules present in the urine carboxylic acid molecules present in the urine 4.4. Then add enzyme-labeled THC-9-carboxylic acid Then add enzyme-labeled THC-9-carboxylic acid

molecules to the urinemolecules to the urine5.5. Any antibodies that did not bind to THC-9-carboxylic Any antibodies that did not bind to THC-9-carboxylic

acid prior to this step (extra antibody molecules), will acid prior to this step (extra antibody molecules), will bind to the enzyme-labeled THC-9-carboxylic acidbind to the enzyme-labeled THC-9-carboxylic acid

6.6. One can now measure the amount of unbound or One can now measure the amount of unbound or unused enzyme-labeled THC-9-carboxylic acid to get unused enzyme-labeled THC-9-carboxylic acid to get a value of THC originally present in the urine.a value of THC originally present in the urine.

Questions for a Criminalist:Questions for a Criminalist:

1.1. Is it blood?Is it blood?

2.2. Is it human blood?Is it human blood?

3.3. If it is human, can it be linked to a If it is human, can it be linked to a suspect / victim and how closely?suspect / victim and how closely?

Color tests to identify human blood

The Benzidine color test was used for years to identify human blood

But benzidine was identified as a carcinogen, so nowadays the phenolphthalein test is performed instead.

The phenolphthalein test is also known as the Kastle-Meyer color test

Both, the benzidine and the Kastle-Meyer test look for the presence of hemoglobin in the sample.

The Kastle-Meyer Test

When blood, phenolphthalein and hydrogen peroxide are mixed, the hemoglobin in the blood will cause the normally colorless phenolphthalein to a bright pink color

This test can yield some false positivesCertain vegetable matter can produce the

bright pink positive (such as potatoes)

Death By Potato?Death By Potato? Although the Although the

Kastle-Meyer test Kastle-Meyer test can give a false can give a false positive in the positive in the presence of certain presence of certain plant matter, it is plant matter, it is unlikely that this unlikely that this plant matter would plant matter would be present at a be present at a crime scene and be crime scene and be mistaken for blood.mistaken for blood.

Other blood detection testsOther blood detection tests

Doctors use Hemastix© strips to detect Doctors use Hemastix© strips to detect blood in urineblood in urine

But these strips can be used at a crime But these strips can be used at a crime scene to detect fresh or dried blood.scene to detect fresh or dried blood.

One simply moistens them with distilled One simply moistens them with distilled water and wipes the bloodstainwater and wipes the bloodstain

A color change to green is a positive A color change to green is a positive indicator of blood indicator of blood

Bayer Bayer Hemastix©Hemastix©

LuminolLuminol LuminolLuminol is a chemical that exhibits is a chemical that exhibits

chemiluminescence, with a striking blue chemiluminescence, with a striking blue glow, when mixed with an appropriate glow, when mixed with an appropriate oxidizing agent. It is a white to slightly oxidizing agent. It is a white to slightly yellow crystalline solid that is soluble in yellow crystalline solid that is soluble in water and most polar organic solvents.water and most polar organic solvents.

Usually, a solution of hydrogen peroxide Usually, a solution of hydrogen peroxide (H(H22OO22) and a hydroxide in water is used as ) and a hydroxide in water is used as the activator.the activator.

In the presence of a catalyst such as an In the presence of a catalyst such as an iron compound, the hydrogen peroxide is iron compound, the hydrogen peroxide is decomposed to form oxygen and water: decomposed to form oxygen and water:

What do you see with What do you see with luminol?luminol?

It glows a bright blue in the dark, when it comes in contact with blood

Advantages of using LuminolAdvantages of using Luminol

Allows one to detect stains that would Allows one to detect stains that would not be ordinarily be visiblenot be ordinarily be visible

Extremely sensitive - can use it in Extremely sensitive - can use it in very dilute concentrationsvery dilute concentrations

This allows the CSI to spray large This allows the CSI to spray large areas with itareas with it

It does not interfere with DNA, so a It does not interfere with DNA, so a CSI can collect samples for DNA CSI can collect samples for DNA analysis even after it was sprayed analysis even after it was sprayed with luminol.with luminol.

Bloody Footprints that were Bloody Footprints that were wipedwiped

DrawbacksDrawbacks

Luminol glows even Luminol glows even in the presence of in the presence of certain other fluids – certain other fluids – semen, feces, bleach, semen, feces, bleach, tonic water, etc.tonic water, etc.

Luminol glows in the Luminol glows in the presence of certain presence of certain vegetable - Mr. vegetable - Mr. Potato head strikes Potato head strikes again!again!

Thanks to Quinine

Crystal TestsCrystal Tests

Less sensitive than color testsLess sensitive than color tests They involve the crystalization of They involve the crystalization of

certain components of blood when certain components of blood when certain chemicals are addedcertain chemicals are added

Takayama test and Teichman test Takayama test and Teichman test Not used as much by CSINot used as much by CSI

So we know it’s blood – but is it So we know it’s blood – but is it human?human?

The Precipitin TestThe Precipitin Test– Rabbits injected with human bloodRabbits injected with human blood– They make antibodies in their serumThey make antibodies in their serum– They are bled and the serum recoveredThey are bled and the serum recovered– The serum is called human antiserum, The serum is called human antiserum,

because it will react to human antigensbecause it will react to human antigens– It will cause coagulation when mixed with It will cause coagulation when mixed with

human bloodhuman blood

Precipitin TestPrecipitin Test

(Human blood)

(Human antiserum made in rabbits)

Gel Diffusion TestGel Diffusion Test

Antigen (blood) and antibodies Antigen (blood) and antibodies (human antiserum from rabbits) are (human antiserum from rabbits) are placed in separate wells in a gel.placed in separate wells in a gel.

They are forced to move towards They are forced to move towards each other.each other.

If they bind to each other, they form If they bind to each other, they form a single line of precipitate in between a single line of precipitate in between the wellsthe wells

This is a positive for human bloodThis is a positive for human blood

DNA TESTDNA TEST

DNA testing is the ultimateDNA testing is the ultimate It has made most other tests for It has made most other tests for

determining if the blood is human, determining if the blood is human, obsoleteobsolete

Analyzing Bloodstain Analyzing Bloodstain PatternsPatterns

1.1. Surface TextureSurface Texture– The harder and less porous the a surface, the The harder and less porous the a surface, the

less spatter resultsless spatter results2.2. Direction of travelDirection of travel

– The pointed end of a bloodstain always faces The pointed end of a bloodstain always faces the direction of travelthe direction of travel

3.3. Impact angleImpact angle– If the strike agle is 45deg then the stain is If the strike agle is 45deg then the stain is

circular. As the strike angle increases, the circular. As the strike angle increases, the stain becomes more and more elongatedstain becomes more and more elongated

4.4. Origin of blood spatterOrigin of blood spatter– If you draw straight lines trhough the long If you draw straight lines trhough the long

axis of several blood stains, the point where axis of several blood stains, the point where the lines converge is the origin of the blood the lines converge is the origin of the blood spatterspatter

Types of Blood Stain Types of Blood Stain PatternsPatterns

1.1. Passive Passive

2.2. ProjectedProjected

3.3. TransferTransfer

1. Passive BloodstainsPassive stains are drops created or formed by the force of gravity acting alone.

This category can be further subdivided to include:

Drops DripsPools

Passive drops on various surfaces

• Blood droplets that strike a hard smooth surface, like a piece of glass, will have little or no distortion around the edge.

• Blood droplets that strike a rough surface have a slightly different appearance. They tend to be distorted, scalloped or spined at the edges.

Passive: Dripped Blood

patterns created by a volume of blood, from same source to target distance (repeated drops onto same spot.)

2. Projected Bloodstains• Projected bloodstains are created when an

exposed blood source is subjected to an action or force, greater than the force of gravity. (Internally or Externally produced)

• The size, shape, and number of resulting stains will depend, primarily, on the amount of force utilized to strike the blood source.

• This category can be further subdivided to include:– Arterial Gush or spurt– Cast-off stains– Impact spatter (medium or high velocity)

Arterial gush or spurt• Bloodstain pattern (s) resulting from blood

exiting the body under pressure from a breached artery.

Cast-off Stains• Blood released or thrown from a blood-bearing object

in motion (Knives, bludgeons)

Projected blood through a syringe

Impact Spatter

• Blood stain patterns created when a blood source receives a blow or force resulting in the random dispersion of smaller drops of blood.

• This category can be further subdivided into; – Low Velocity– Medium Velocity– High Velocity

Low Velocity Impact Spatter

•Gravitational pull up to 5 feet/sec. •Relatively large stains 4mm in size and greater

Medium Velocity Impact Spatter

• Force of 5 to 25 feet/sec. • Preponderant stain size 1 to 4mm in size

High Velocity Impact SpatterForce of 100 feet/sec. and greaterPreponderant stain size 1mm in size and smaller Mist like appearance

3. Transfer Bloodstains

• A transfer bloodstain is created when a wet, bloody surface comes in contact with a secondary surface.

• A recognizable image of all or a portion of the original surface may be observed in the pattern,as in the case of a bloody hand or footwear.

THE ENDTHE END