1. Chapter 10 Forensic Serology

2. 6.2 - Blood as Evidence
While examining blood from a crime scene, the criminalist must answer:
Is it blood?
2. Is it human blood (or what species)?
3. If the blood is of human origin, how
closely can it be associated to a
particular individual?

3. What is the most common blood typing system used
What is the most common blood typing system used? The A-B-O blood typing system
Karl Landsteiner – Discovered the A-B-O blood typing system.
*There are other blood typing systems, the ABO system is just the most immunogenic.
Why is knowing your blood type so important?
Leonard Landois – Observed clumping of blood when two samples were mixed.

5. SEROLOGY: The study of antigen-antibody reactions.
GENERAL DEFINITIONS
SEROLOGY: The study of antigen-antibody reactions.
IMMUNOASSAY: All aspects of the immune system
*Includes methods to measure antigen-antibody interactions.
Blood: A complex mixture of cells, enzymes, proteins, and inorganic substances.
Plasma is the fluid portion of blood.
-Mostly water; Is 55% of blood content.
-Suspended in plasma are solid materials, 45% of blood.
*Red blood cells – erythrocytes
*White blood cells – leukocytes
*Platelets

6. Liquid Components of Blood
Plasma: The liquid portion of blood that contains…
*nutrients (glucose, amino acids, lipids, salts, fatty acids)
*Ischemia: when blood supply is cut off from an organ;
Could be detrimental to the organ.
Blood Clot- Fibrins (a protein) traps & enmeshes the red blood cells.
Serum: Yellow liquid that separates from the plasma when the clotting solids are removed.

7. CELLULAR COMPONENTS OF BLOOD
Red Blood Cells – erythrocytes
*Transport oxygen & carbon dioxide
*Although cells, RBC’s do not have a nucleus
no nucleus = no DNA
White Blood Cells – leucocytes
*Neutrophils, B-cells, T-cells, monocytes, natural killer cells (NK’s)
Platelets – thrombocytes
*Small, disc-shaped cells; Involved in blood-clotting &
assist in forming fibrin.

8. Platelets

9. Presumptive Blood Tests
IS IT BLOOD???
Presumptive Blood Tests
Color Tests: Guiacum test (early test), Kastle-Meyer Test
2) Luminol & Fluorescein
3) Microcrystalline Tests: Takayama Test

10. Guiacum Test
Presumptive color test for blood. *A positive test for blood results in a BLUE color change. *Blood reacts with alpha giaiaconic acid.

11. Kastle-Meyer Color Test: Uses the indicator phenolphthalein and hydrogen peroxide to react with hemoglobin releasing peroxidase enzymes to produce a pink color.
*False positive – potatoes, horseradish. However, in a criminal context, a positive result is highly indicative of the presence of blood.

12. Luminol: Fluoreses when iron from hemoglobin catalyzes a chemical reaction.
*Can spray a large area.
*Must be a darkened area.
*Very sensitive – detects blood diluted to 1 in 100,000.
*Do not interfere with DNA testing of blood samples.
Fluorescein – Req’s UV light to fluoresce

13. TakayamaTest
Pyridine & glucose are added to blood result in characteristic crystals with hemoglobin derivatives.
Less sensitive
Susceptible to interference by other substances in the blood
Is considered to be a confirmation test for blood.

14. Question #2: Is it human blood?
To determine if blood is human, a reaction between antigens and antibodies must be used. Immunoassay: Process in which specific antibodies are used to identify biological samples.

15. QUES. #2 – Is it human blood?
Precipitin Test: An antigen-antibody reaction between a blood sample and an antiserum.
*’Anti-human serum’ coagulates human blood.
Very sensitive test
can test small amounts as old as years and longer (mummies 4-5 thousand years old have provided positive reactions)
Human blood stains washed in water have also provided positive results

16. Human blood gives a precipitin band with sensitized rabbit serum
Human Anti-serum
from rabbit

17. Gel Diffusion Method of precipitin test
Antigen and antibody are added to their respective wells
Antigen and antibody are being moved toward each other
Antigen and antibody have formed a
visible precipitin line in the gel between
the wells, if of human origin

18. Immunoassay Techniques:
(EMIT & Monoclonal Antibodies)
Using the antigen-antibody reaction for other uses besides blood typing.
Drugs: Detected in blood or urine
*Antibodies against a drug are produced (since
they don’t occur naturally).
*Drug is attached to a protein and injected
into a rabbit.
*The rabbit produces antibodies against the drug.
*The antibodies are collected from the
rabbit’s blood sample and reproduced.

19. Immunoassay Techniques: EMIT
EMIT: Enzyme-Multiplied Immunoassay Technique
*Adding antibodies for a drug to a urine sample
*Add the ‘mixture’ to a chemically labeled drug
sample. Labeled drug will bond with the
antibodies if drugs aren’t already in the urine.
If drugs are present in the urine sample…
*There will be a high concentration of labeled
drug in the urine.
If drugs are not present in the urine sample…
*There is a low concentration of labeled drug
in the urine.

20. ELISA TEST

21. Immunoassay Techniques: Monoclonal Antibodies
Monoclonal Antibodies: Identical antibodies that interact with a single antigen site.
Polyclonal Antibodies: A series of antibodies produced responding to a variety of different sites on the antigen.
*One specific antibody for one specific
antigen site is difficult to create.
How can a monoclonal antibody be created?

22. How to make monoclonal antibodies-
*Inject a mouse with an antigen. It’s spleen will start making antibodies.
*Spleen cells containing the antibody are collected from the mouse & hybridized (attached to) fast-growing blood cancer cells.
*These hybridoma cells are allowed to multiply and result in a high yield of the antibody.
Antibodies are being used in forensics for…
*Drug tests, seminal material
Also, as a cancer treatment

23. Unrelated to this function, there are antigens on the surface of the RBC.
Antigen: A substance (usually protein) stimulates the body to produce antibodies against it.
*Antigens on RBC designate the blood-type of a person.
There are 29 known blood antigen (blood typing) systems. The A-B-O and Rh systems are the most important.
*A-B-O = Blood type
*Rh factor (D antigen) = + or –
*For every antigen, there exists a specific antibody.

24. Red Blood cell surface:
* The ABO Blood System *
Blood type
(genotype
Type A
(AA, AO)
Type B
(BB, BO)
Type AB
(AB)
Type O
(OO)
Red Blood cell surface:
Antigens present
A antigen
B antigen
No antigens
Antibodies
Present in serum/
plasma
A & B antigens
NONE
Anti-B antibodies
Anti-A antibodies
No antibodies
A & B antibodies

25. Blood Types & Antibodies Present
The antiserum specific for an antigen will attached to antigens and create a network of cells. Causes clumping of cells- agglutination.

27. What blood type is shown?

28. Genotype (DNA-Alleles) Blood Type (Phenotype)
Rh Factor – D antigen
* A protein (also found in Rhesus Monkeys) used in blood typing
*Present = (+)
*Absent = (-)
*Population breakdown:
85% is Rh +
15% is Rh –
*Rh+ gene is dominant
Genotype (DNA-Alleles)
Blood Type (Phenotype)
(+ , -) or (+ , +)
Rh +
(- , -)
Rh -

29. Blood Donor Table A A, AB A, O B B, AB B, O AB AB, A, B, O O
Blood Type
Can Donate To
Can Receive From A
A, AB
A, O B
B, AB
B, O AB
AB, A, B, O O
A, B, AB, O
The blood typing tutorial link below may be helpful for further understanding.

30. Principles of Heredity
Genes: Microscopic unit of hereditary material Genes are located on chromosomes – threadlike bodies in the nucleus of every body cell. *Human cells contain 46 chromosomes (23 pairs) *Human reproductive cells contain 23 individual chromosomes. Egg: Female reprodctive cell Sperm: Male reproductive cell Zygote: Cell that arises from the union of egg & sperm.

31. Principles of Heredity
* Allele- Alternate forms of genes that influence a given characteristic and are aligned with one another on a chromosome pair
There are 3 alleles for the ABO blood typing system:
A, B & O - A & B are co-dominant, O is recessive
* Homozygous- having 2 identical allelic genes on two corresponding positions on a pair of chromosomes
AA, BB, OO
* Heterozygous-having 2 different allelic genes on two corresponding positions on a pair of chromosomes
AO, AB, BO

32. *Genotype (DNA-Alleles)
* Genotype: A particular combination of genes present in the cells of an individual
* Phenotype: The physical manifestation of a genetic trait such as shape, color, and blood type.
*Genotype (DNA-Alleles)
*Blood Type
(Phenotype)
AO or AA
A blood type (~42%) AB
AB blood type (~3%)
BO or BB
B blood type (~12%) OO
O blood type (~43%)

33. A B B A B B B O A O B O

34. Stain Patterns of Blood
6.3 - BLOOD SPATTER
Stain Patterns of Blood
The location, distribution, and appearance of bloodstains and spatters may be useful for interpreting and reconstructing the events that resulted in blood loss.
A variety of factors influence blood spatter patterns.
Categories of Bloodstains
(Passive, Transfer, Active)

35. 1) PASSIVE Bloodstains
Passive Bloodstains are drops created or formed by the force of gravity acting alone.
*This category can be further subdivided to include;
Drops
Drip patterns
Pools
Clots
*Blood travels with low velocity
*When gravity pulls a blood drop straight down, the resulting shape is very close to a perfect circle.

36. This category can be further subdivided to include;
2) Active 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 Spurt / Gush
Bloodstain pattern(s) resulting from blood exiting the body under pressure from a breached artery

37. Active Bloodstains, cont.
VELOCITY OF BLOOD Velocity of traveling blood changes due to forces on the blood.
Low Velocity: Gravitational pull up to 5 feet/sec. Relatively large stains 4mm in size and greater (circular drop shape)
Medium Velocity: Force of 5 to 25
feet/sec. Stain size 1 – 4 mm.
High Velocity: Force of
100 feet/sec. and greater Stain size 1mm in size and
Smaller. Mist like appearance

38. Active Bloodstain, cont. - Arterial wound AKA – Arterial spurt, gush
*Blood is under the force of a person’s blood pressure due to the heart still beating.
*Wave-like pattern / Inverted V

39. Active Bloodstain, cont. - Cast off Pattern
When blood is flung from an object.
*Blood cast off of an object travels in a straight line.
*Helpful in determining the direction of movement of the weapon * the minimum number of impacts that occurred in the assault (one line of drops will be present for each swing/impact)

40. DIRECTIONALITY OF BLOODSTAINS
When a droplet of blood strikes a surface perpendicular (90 degrees) the resulting bloodstain will be circular (length and width of the stain will be equal).
Blood that strikes a surface at an angle less than 90 degrees will be elongated or have a tear drop shape.
*Directionality is usually obvious
as the pointed end of the
bloodstain (tail) will always point in the direction of travel.

41. Angle of impact: can be determined
by measuring the degree of circular distortion.
In your calculator:
Sin-1 W/L

42. IMPACT ANGLE DETERMINATION
ANGLE of IMPACT is the acute angle formed between the direction of the blood drop and the plane of the surface it strikes

43. AREA OF ORIGIN
The common point, on a 2 dimensional surface, over which the directionality of several bloodstains can be retraced.
Once the directionality of a group of stains has been determined, it's possible to determine a two dimensional point or area for the group of stains.

46. Transfer bloodstains can be further subdivided into;
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.
Transfer bloodstains can be further subdivided into;
Contact bleeding
Swipe or Smear
Wipe
Smudge

47. LARGE VOLUMES OF BLOOD
(patterns created by same volume of blood, from same source to target distance)
"Dripped Blood"
"Spilled Blood"
"Projected Blood" (through syringe)

48. 6.4 – SEROLOGY & OTHER BIOLOGICAL FLUIDS
Saliva – A mixture containing approx. 99% water with small amounts of proteins, enzymes, mucus, electrolytes, and other components.
*Secreted by glands located in the mouth
*Aids in the digestion
Forensics Uses – Supports allegations of inappropriate sexual contact. Can be recovered from a suspect’s body as well as eating/drinking utensils, cigarettes, stamps.
*Detection of drugs
Presumptive Tests – Identify presence of amylase or squamous epithelial cells shed by the inside of our mouth.
*False positive for amylase is possible
*Cells identified microscopically

49. 6.4 cont. - Semen Identification
*Semi-fluid necessary to carry sperm
*Cells, proteins, amino acids, hormones, carbohydrates, and a variety of other inorganic and organic compounds
FORENSIC APPLICATIONS
*Sexual assault cases
*Microscopically identified
*Drugs, alcohol, disease, and vasectomy, can drastically
decrease the sperm count

50. Presumptive Tests –
SAP – Seminal Acid Phosphatase
*Enzyme found in high levels & present when sperm are absent
*Turns blue in the presence of semen
Confirmation Tests –
Microscopy
Identification of PSA or p30 (prostate specific antigen)
Y-Detect: Detects the DNA of male sperm cells
*Very sensitive, highly specific, all human cells are testable
Rapid Stain Identification (RSID): Uses monoclonal antibodies specific for seminal fluid that don’t react with other body fluids.
*Antibodies are specific for semenogelin antigens)

51. 6.4 - Urine Urine eliminates waste and regulates ion concentration
*Composed mostly of water & salts; Formed in the kidneys
Forensic Applications – Search/identify qualitative presence of drugs, poisons, alcohol.
*Presumptive tests look at the conc. of urea & creatinine
Other Body Fluids
Sweat – Contains hormones & proteins but can also be used to determine blood type if the person is a ‘secretor’
Bile – Used to detect drugs, poisons in post mortem samples
Vitreous humor – Fluid behind eye lens; post mortem determination of disease, detection of drugs and poisons, and in the estimation of the post mortem interval (PMI).