1. Chapter 12 Serology Students will understand the process of blood typing Students will identify characteristics of blood stains

2. I. Serology A. General Concepts A. General Concepts –1. Karl Landsteiner discovered blood had different “types”  a) Was the foundation work for A-B-O –2. Blood “factors” were genetic –3. DNA testing of blood has aided in the linking of suspects to crimes

4. I. Serology B. Nature of Blood B. Nature of Blood –1. Antigens and antibodies  a) Blood refers to a complex mixture of cells, enzymes, proteins, and inorganic substances  b) Plasma is the fluid portion –i) Mostly water at approximately 55% of blood content –ii) Solid suspensions are RBC’s, WBC’s, and platelets –iii) Clotting occurs due to the protein fibrin- traps red blood cells  Crack or remove a clot and there is a pale-yellow liquid called serum (can be sticky!)

6. I. Serology  c) Antigens are characteristic chemical structures on the RBC’s –i) Grouped into systems  Currently there are 15 known systems  A-B-O and Rh are most important –ii) Type A has the A antigen (or protein) –iii) Type B has the B antigen –iv) Type AB has both antigens –v) Type O has neither antigen –vi) The Rh factor is also called the D antigen  Is identified as (+) or (-)

7. I. Serology  d) Serum contains antibodies –i) For blood typing, the fundamental principle is that for every antigen there is an antibody  For example) Anti-A is an antibody for antigen A –ii) The serum containing the antibody is called antiserum  Meaning a serum that reacts against something (antigens) –iii) Antibodies are specific  Contain 2 active sites (bivalent)  Can create cross linked cells seen as clumping or agglutination

8. I. Serology Blood Type Antigens on Red Blood Cells Antibodies in Serum AAAnti-B BBAnti-A ABAB Neither anti-A nor anti-B O Neither A nor B Both anti-A and anti-B

11. I. Serology B. Nature of Blood B. Nature of Blood –2. Blood Typing  a) Serology is used to describe the study of blood and the tests run to determine specific blood types  b) Testing requires unknown blood types tested against the 3 antiserums –3. Immunoassay  a) One antigen- one antibody (lock-n-key)  b) Used to determine if drugs are present in urine and blood –i) Developed by introducing drugs into rabbits creating a specific antibody for that drug –ii) The blood serum is removed from the rabbit

12. I. Serology  c) Enzyme-Multiplied Immunoassay Technique (EMIT) –i) A technique used to detect drugs –ii) High speed and highly sensitive detection in urine  Can determine amount of drug in urine (ex. Methadone)  Used primarily for marijuana (THC)  Body converts THC into THC-a-carboxylic acid (in very small amounts, but are detected)  Positive results are attainable up to 10 days after use

13. Enzyme-Multiplied Immunoassay Technique (EMIT)

14. I. Serology  d) Radioimmunoassay –i) Uses drugs that are “tagged” with radioactive markers  e) Immunoassays are not 100% accurate –i) Drugs with similar metabolic structures can give positive results (ex. Poppy seeds) –ii) Must be confirmed C. Characteristics of Blood Stains C. Characteristics of Blood Stains –1. General Concepts  a) must answer the following –i) Is it blood? –ii) From what species did it come from? –iii) If human, does it come from an individual involved in the crime scene?

15. I. Serology C. Characteristics of Blood Stains C. Characteristics of Blood Stains –2. Types of testing procedures  a) Preliminary Color Test –i) Benzidine color test (carcinogen) –ii) Phenolphthalein (Kastle-Meyer color test)  Contain peroxidase activity (blood)  When mixed with blood the chemical reaction causes a deep pink color  Can have false positives with some vegetables (ex. potatoes and horseradishes)

16. I. Serology C. Characteristics of Blood Stains C. Characteristics of Blood Stains –2. Types of testing procedures (cont)  a) Preliminary Color Test –iii) Luminal  Production of light not color  Quickly screen a large area with spray  Extremely sensitive –iv) Microcrystalline test  Characteristic crystals when introduced with hemoglobin are formed

17. Benzidine Color Test

18. Phenolphthalein (Kastle-Meyer Color Test)

20. Luminal

22. Microcrystalline Test

24. CocaineMethamphetamine

25. I. Serology C. Characteristics of Blood Stains C. Characteristics of Blood Stains –3. Secondary Testing Procedures  a) Precipitin determines if the blood stain is human –i) Rabbits are injected, if it is human antibodies are formed causing agglutinations  Create human antiserum and is used to test the blood

26. I. Serology C. Characteristics of Blood Stains C. Characteristics of Blood Stains –3. Secondary Testing Procedures (cont)  b) Gel diffusion methods are used to determine if blood is human –Antibodies and antigens will move toward each other on an agar plate –Very sensitive test –Very little blood is needed to run the test

27. I. Serology C. Characteristics of Blood Stains C. Characteristics of Blood Stains –3. Secondary Testing Procedures (cont)  c) Typing dried stains –1. Absorption-elution technique  a) Four step process  Step 1: Antiserum is added to blood stain  Step 2: Un-reacted serum is washed off  Step 3: Elution breaks apart the serum and the antigen (heated to 56°C)  Step 4: Eluted antibodies are combined with known blood types, looking for agglutinations

28. I. Serology C. Characteristics of Blood Stains C. Characteristics of Blood Stains –3. Secondary Testing Procedures (cont)  c) Typing dried stains –2. Secretors  Blood-typing antigens are found in high concentration in most body fluids  (tab) Includes saliva, semen, vaginal fluids, and gastric juices

29. I. Serology C. Characteristics of Blood Stains C. Characteristics of Blood Stains –3. Secondary Testing Procedures (cont)  d) Typing blood enzymes and other proteins –enzymes are specialized proteins  Some are polymorphic  (tab) Exist in different forms  (tab) Separated into iso-enzymes  (tab) PGM (phosphoglucomutase) is an example  (tab) Separate the iso-enzyme by gel electrophoresis

30. I. Serology C. Characteristics of Blood Stains C. Characteristics of Blood Stains –3. Secondary Testing Procedures (cont)  d) Typing blood enzymes and other proteins (cont) –enzymes are specialized proteins (cont)  Some are polymorphic  (tab) Individuals have variations in the iso-enzyme  (tab) With increased technology, PGM can be further separated  (tab) There are now 10 PGM iso-enzymes and their combinations in the population can be used as identifiers

31. I. Serology C. Characteristics of Blood Stains C. Characteristics of Blood Stains –3. Secondary Testing Procedures (cont)  d) Typing blood enzymes and other proteins –enzymes are specialized proteins (cont)  Used to individualized blood samples  DNA technology has slowed research into this method

32. I. Serology C. Characteristics of Blood Stains C. Characteristics of Blood Stains –3. Secondary Testing Procedures (cont)  d) Typing blood enzymes and other proteins –Only use the blood enzymes resistant to damage due to desiccation –The more proteins identified, the smaller the possible population with those protein combinations

33. Blood Factor Abbreviation Adenosine deaminase ADA Adenylate kinase AK Carbonic anhydrase II CA II Erythrocyte acid phosphate EAP Esterase D EsD Glucose-6-Phosphate dehydrogenase G6PD Glyoxylase I GLO I Group-specific component Gc HaptoglobinHp Peptidase A Pep A PhosphoglucomutasePGM 6-Phosphogluconate dehydrogenase 6PGD TransferrinTf Blood Enzymes and Proteins Used to Discriminate Bloodstains

34. Grouping System Type Frequency (approx.) ABO043% ADA190% AK191% EAPBA43% EsD179% GLO I 2 – 1 52% Gc150% Hp 49% PGM 1 + 1 + 41% 6PGDA96% TfCC98% Possible groupings and frequency math Ex) O + AK + EsD + Hp + Gc 0.43 x 0.91 x 0.79 x 0.49 x 0.50

35. I. Serology D. Stain Patterns D. Stain Patterns –1. General Concepts  a) Blood observations can help reconstruct crime scenes  b) Studies have developed patterns related to dropping distance and angle of impact –i) Also consider surface texture  Like surfaces must be used during comparisons –ii) Consider travel direction, the pointed end faces the travel direction –iii) Consider impact angle, measure the degree of circular distortion  90° gives nearly circular surface  Decreasing angles give elongation to the blood stain

36. Passive Blood Patterns

37. Directional Blood Pattern

38. Determining Blood Pattern

39. Patterned Blood Smear

41. Projection Blood Pattern

42. Blood Patterns

43. I. Serology iv) By accurately measuring the length and width of a bloodstain, the impact angle can be calculated using the SIN formula below iv) By accurately measuring the length and width of a bloodstain, the impact angle can be calculated using the SIN formula below (indent) Example (indent) Example (indent) SIN < = Width (a) Length (c) (indent) Width (a) [1.5cm] = SIN < (indent) Length (c) [3.0cm] 0.5 = SIN < < = 30 degrees

44. I. Serology D. Stain Patterns or Blood Spatter D. Stain Patterns or Blood Spatter –1. Generally there are two types of blood spatter  a. Medium Force (velocity) impact spatter –Also called projected spatter –Is produced with more energy or force than the force of gravity –The force of impact causes the blood to break into smaller size spatters (relative to the applied force) –Usually scene with blunt force trauma, stabbings, and secondary spatters

45. I. Serology D. Stain Patterns or Blood Spatter (cont) D. Stain Patterns or Blood Spatter (cont) –1. Generally there are two types of blood spatter (cont)  a. Medium Force (velocity) impact spatter (cont) –Produced when the majority of larger drops are broken into smaller spatters –Tend to be a diameter of 2-4 mm in diameter –The force is greater than 25 ft/s or 7.62 m/s

46. I. Serology D. Stain Patterns or Blood Spatter (cont) D. Stain Patterns or Blood Spatter (cont) –1. Generally there are two types of blood spatter (cont)  a. High Force (velocity) impact spatter –Measures less than 2mm in diameter –Must have a force greater than 100 ft/s or 30.48 m/s –Typically associated with gunshots, explosions, and high speed collisions –Appears ‘mist-like’ after impact –Can be hard to see with the naked eye

47. I. Serology D. Stain Patterns (cont) D. Stain Patterns (cont) –2. POINT OF CONVERGENCE AND ORIGIN DETERMINATION   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.   By drawing a line through the long axis of a group of bloodstains the point of convergence can be determined. Where the lines of the group of stains intersect one another the convergence point can be established.

48. I. Serology

50. Example of Point of Origin

52. I. Serology  Determine the origin of the blood splatter, draw straight lines through the long axis of several individual blood stains –Converging point determines POI (point of origin) E. Characterization of Semen E. Characterization of Semen –1. General Concepts  a) Males release 2.5 to 6 mls of seminal fluid  b) Approximately 100 million sperm  c) Must locate the stain –i) Acid Phosphate Color Test  Checks for acid phosphate  Uses sodium alpha naphthylphosphate and fast-blue B dye, along with 4-methyl umbelliferyl phosphate (MUP)  Fluorescence under UV light

53. I. Serology  d) Perform tests on stain after located  e) Sperm can be difficult to test –i) Become brittle –ii) Bind tightly to cloth –iii) Many sex crimes are committed by men with low sperm counts (oligospermia) or have no sperm count (aspermia)  f) When sperm is not present, yet the investigator believes the material is semen –i) Can test for the presence of p30 (Prostate Specific Antigen)  Unique to seminal plasma  Can be discovered by an Anti-serum from rabbits

54. I. Serology F. Collection of Rape Evidence F. Collection of Rape Evidence –1. General Information  a) Bag all outer and undergarments in separate bags  b) Place clean sheet on the floor, remove shoes, remove clothes, bag clothes, fold up sheet  c) Stained clothing should NOT have folds, material may rub off  d) Must put victim through a medical exam –2. Medical Exam Procedures  a) Pubic combing/ add pubic hair controls  b) Vaginal swabs and smear  c) Rectal swab and smear

55. I. Serology  d) Oral swab and smear  e) Pulled head hairs  f) Blood samples  g) Fingernail scrapings –3. Materials from suspects  a) All clothing  b) Pubic hair combing  c) Pulled head and pubic hair controls  d) Blood samples –4. Survivorship sperm  a) Live sperm can be found up to 6 hours after crime (living person)  b) Dead sperm can be found up to 3 days after intercourse (sometimes 6 days)