1. Serology Chapter 6

2. Three main questions Is this blood?Is this blood? Is this blood human?Is this blood human? What biochemical markers are found in the blood?What biochemical markers are found in the blood?

3. More than just ID Blood at a crime scene can be analyzed byBlood at a crime scene can be analyzed by Composition of the bloodComposition of the blood Patterns of blood samplePatterns of blood sample Location of blood sampleLocation of blood sample To tell a significant amount of informationTo tell a significant amount of information

4. Detection of Blood Odor, color, and texture are not enough to detect human bloodOdor, color, and texture are not enough to detect human blood Dried blood look’s nothing like bloodDried blood look’s nothing like blood Guaiacum test: plant extract turned blue when in contact of bloodGuaiacum test: plant extract turned blue when in contact of blood Not reliableNot reliable Human = animal bloodHuman = animal blood

5. Detection of Blood Kastle-Meyer testKastle-Meyer test Uses phenolphthalein (indicator of acid)Uses phenolphthalein (indicator of acid) Mixed with H2O2 and blood, changes from colorless to pinkMixed with H2O2 and blood, changes from colorless to pink False positivesFalse positives Animal = humanAnimal = human

6. 2 Stories of Blood Discoveries Louis Pasteur (1879)Louis Pasteur (1879) discovered immunity to diseases discovered immunity to diseases Injected chickens with choleraInjected chickens with cholera Chickens got sick, but recoveredChickens got sick, but recovered Never became infected againNever became infected again Worked with anthrax and rabiesWorked with anthrax and rabies

7. 2 stories of blood discoveries Edward Jenner (1796)Edward Jenner (1796) Used cowpox injections to produce immunity for smallpox diseaseUsed cowpox injections to produce immunity for smallpox disease Came up with “Vaccination” from the Latin name of the smallpox virus (vaccinia)Came up with “Vaccination” from the Latin name of the smallpox virus (vaccinia)

8. Detect HUMAN blood Uhlenhuth Serum-based test (1900)Uhlenhuth Serum-based test (1900) First test to identify HUMAN bloodFirst test to identify HUMAN blood Detection of specific other species of animal blood has been since discoveredDetection of specific other species of animal blood has been since discovered

9. Check Point How can we determine if the substance is blood?How can we determine if the substance is blood? How can we determine if the substance is Human blood?How can we determine if the substance is Human blood? Next question: how can we determine WHO’S blood it is?Next question: how can we determine WHO’S blood it is? Guaiacum test & Kastle-Meyer testGuaiacum test & Kastle-Meyer test Uhlenhutch serum- based testUhlenhutch serum- based test

10. Did you know? The adult human body contains 5.5 liters of blood. The heart pumps nearly 300 liters every minute!The adult human body contains 5.5 liters of blood. The heart pumps nearly 300 liters every minute!

11. Who’s blood it is? Discovery by successful and unsuccessful transfusions…Discovery by successful and unsuccessful transfusions… In Medieval times, people would replenish blood supply on humansIn Medieval times, people would replenish blood supply on humans Sometime person died, sometime livedSometime person died, sometime lived

12. Fatal Transfusions? (1875) Observed that when human blood mixed, often clumping resulted…led to death(1875) Observed that when human blood mixed, often clumping resulted…led to death (1901) Karl Landsteiner discovered how to avoid this(1901) Karl Landsteiner discovered how to avoid this Found ABO systemFound ABO system

13. How can Blood type be determined? Presence or absence of cell- surface proteinsPresence or absence of cell- surface proteins Anti-body reaction testAnti-body reaction test

14. Composition of Blood Blood = Tissue!Blood = Tissue! Red blood cellsRed blood cells White blood cellsWhite blood cells PlateletsPlatelets Suspended in plasmaSuspended in plasma Carries proteins: antibodies and hormones, clotting factors, nutrients, amino acids, salts, mineralsCarries proteins: antibodies and hormones, clotting factors, nutrients, amino acids, salts, minerals

15. Composition of Blood The most of our blood consists of plasma, then the red blood cells follow. White blood cells makes up the least amount of our bloodThe most of our blood consists of plasma, then the red blood cells follow. White blood cells makes up the least amount of our blood

16. Blood Cells Red blood cells (erthrocytes) carry oxygen and CO2Red blood cells (erthrocytes) carry oxygen and CO2 Hemoglobin contains iron which bind to O2 in lungs and carries it to cells in all tissues of the bodyHemoglobin contains iron which bind to O2 in lungs and carries it to cells in all tissues of the body

17. Blood Cells White blood cells (leukocytes) fight disease and foreign invadersWhite blood cells (leukocytes) fight disease and foreign invaders Platelets (thromocytes) aid in blood clotting and repair of damaged blood vesselsPlatelets (thromocytes) aid in blood clotting and repair of damaged blood vessels

18. Immune System Our bodies can determine “self” and “non-self” particlesOur bodies can determine “self” and “non-self” particles Recognize viruses, bacteria, and parasitesRecognize viruses, bacteria, and parasites When found, white blood cells engulf the particle and digest itWhen found, white blood cells engulf the particle and digest it

19. Immune Response Antibodies are secreted from other white blood cellsAntibodies are secreted from other white blood cells **White blood cells are the only blood cell that has a nucleus**White blood cells are the only blood cell that has a nucleus Only blood cell that is a source for DNA and used for DNA profilingOnly blood cell that is a source for DNA and used for DNA profiling

20. Immune Response

21. History of DNA Profiling (1982)- white blood cells were first used as a source of DNA profiling(1982)- white blood cells were first used as a source of DNA profiling Now- DNA profiling and DNA fingerprintingNow- DNA profiling and DNA fingerprinting Used in the Innocence Project to help free inmatesUsed in the Innocence Project to help free inmates

22. Blood Typing Checkpoint: Who was the founder of the ABO blood typing system?Checkpoint: Who was the founder of the ABO blood typing system? Karl Landsteiner (1900)Karl Landsteiner (1900) Is blood typing class evidence or individual evidence?Is blood typing class evidence or individual evidence? Class evidenceClass evidence Because many people share the same typeBecause many people share the same type

23. Cell-Surface Proteins The presence/absence of proteins within the cell or plasma membranes of red blood cells determine blood typeThe presence/absence of proteins within the cell or plasma membranes of red blood cells determine blood type An antibody reaction test is used to identify each blood typeAn antibody reaction test is used to identify each blood type Percentage in U.S. population of blood types:Percentage in U.S. population of blood types: Type OType AType BType AB 43%42%12%3%

24. ABO System-Saves Lives

25. ABO SYSTEM

26. Rh Factor Alex Weiner (1940) worked with Rhesus monkeysAlex Weiner (1940) worked with Rhesus monkeys Found another protein in red blood cellsFound another protein in red blood cells 85% of human population of this protein85% of human population of this protein Rh+ (have protein)Rh+ (have protein) Rh- (no protein)Rh- (no protein)

27. Naming of Blood Types By what characteristic is the blood type of an individual determined?By what characteristic is the blood type of an individual determined? By the presence or absence of AB and Rh proteinsBy the presence or absence of AB and Rh proteins Discovered through unsuccessful blood transfusionsDiscovered through unsuccessful blood transfusions If received blood protein foreign to them- antibodies will cause blood to clot…deadlyIf received blood protein foreign to them- antibodies will cause blood to clot…deadly

28. Antibodies B-lymphocytes secrete antibodiesB-lymphocytes secrete antibodies Y-ShapedY-Shaped Bind to molecular shape of an antigen (foreign particle)Bind to molecular shape of an antigen (foreign particle) Binding site @ tip of Y-shaped moleculeBinding site @ tip of Y-shaped molecule

29. Antigen-Antibody response When a foreign invader is recognized by the immune system…attack!When a foreign invader is recognized by the immune system…attack! Antigen: viruses, bacteria, red blood cell proteins for another blood type (transfusion)Antigen: viruses, bacteria, red blood cell proteins for another blood type (transfusion) 1.B lymphocytes makes specific antibodies against that protein 2.Phagocytes engulf the invader

30. Agglutination CLUMPINGCLUMPING When each arm of Y shaped antibody attaches to red blood cellsWhen each arm of Y shaped antibody attaches to red blood cells Make cease blood flowMake cease blood flow Why is this dangerous?Why is this dangerous? Red blood carries oxygen or eliminates CO2… if body does not get this, person will dieRed blood carries oxygen or eliminates CO2… if body does not get this, person will die

31. Check Point What are the 4 components of blood?What are the 4 components of blood? Red blood cells, white blood cells, platelets, and plasma Red blood cells, white blood cells, platelets, and plasma What are the 4 types of blood?What are the 4 types of blood? O, A, B, AB O, A, B, AB What percentage of blood do each of the types account for in the U.S. population?What percentage of blood do each of the types account for in the U.S. population? 43%, 42%, 12%, 3% respectively 43%, 42%, 12%, 3% respectively

32. Blood Transfusions Who can give you blood? People with TYPE O blood are called Universal Donors, because they can give blood to any blood type. People with TYPE AB blood are called Universal Recipients, because they can receive any blood type. Rh +  Can receive + or - Rh -  Can only receive - Universal Donor Universal Recipient

33. Microscopic Views Bird Blood Cat Blood Dog Blood Fish Blood Frog Blood Snake Blood Human Blood Horse Blood

34. Blood Typing Tests 3 separate tests are performed!3 separate tests are performed! 1.Blood is mixed with antibodies that bind to A protein 2.Blood is mixed with antibodies that bind to B protein 3.Blood is tested with the Rh protein What does it mean if the blood clumps with the first test?What does it mean if the blood clumps with the first test? Blood contains A proteins on cellsBlood contains A proteins on cells What does it mean if the blood clumps with the 2 nd test?What does it mean if the blood clumps with the 2 nd test? Blood contains B proteins on cellsBlood contains B proteins on cells

35. Presumptive vs Confirmatory What did we conclude about our tests we completed yesterday?What did we conclude about our tests we completed yesterday? Recall the false positives that resulted with testing horseradish…Recall the false positives that resulted with testing horseradish… Did we have a definite detection of blood?Did we have a definite detection of blood?

36. Presumptive Test We completed presumptive tests…We completed presumptive tests… This color test indicated that a stain was blood- but not what kind of bloodThis color test indicated that a stain was blood- but not what kind of blood This test also gave a false positive result…This test also gave a false positive result…

37. Confirmatory Test By the name…what would you expect this test would result in?By the name…what would you expect this test would result in? Lack sensitivityLack sensitivity Large amount of the substance must be available for the test to be positiveLarge amount of the substance must be available for the test to be positive Performed usually in a laboratory- more equipment and more controlledPerformed usually in a laboratory- more equipment and more controlled

38. Presumptive vs Confirmatory What would be your in distinction between these two test… discuss the differences with your partnerWhat would be your in distinction between these two test… discuss the differences with your partner

39. Compare PresumptiveConfirmatory Highly sensitive to but not specific for a particular substance Lack sensitivity; They test positive for substance in question and ONLY that substance

40. Studies in serology What materials do serologist study and analyze?What materials do serologist study and analyze? What are the body fluids possible to be studied?What are the body fluids possible to be studied?

41. Presumptive Tests for BLOOD React with hemoglobinReact with hemoglobin Color reactors (clear to pink)Color reactors (clear to pink) Light @ wavelength emittedLight @ wavelength emitted Luminol (chemiiluminescence or fluorescence)Luminol (chemiiluminescence or fluorescence) phenolphthaleinphenolphthalein

42. Presumptive tests for blood Luminol (chemical that emitted light as a by product of a reaction on blood stains) Luminol (chemical that emitted light as a by product of a reaction on blood stains) Fluorescein (emitted light as energy loss at a longer wavelength than it is illuminated with) Fluorescein (emitted light as energy loss at a longer wavelength than it is illuminated with) Must be viewed in the dark or with alternate light source (ALS)Must be viewed in the dark or with alternate light source (ALS) Photographic techniques capture the imagesPhotographic techniques capture the images Emitted light is temporaryEmitted light is temporary Interfer with subsequent testInterfer with subsequent test

43. Confirmatory Tests for blood Use of crystal formation by heat and chemicals- observed under microscopeUse of crystal formation by heat and chemicals- observed under microscope Takayama test (or hemochromagen test) Takayama test (or hemochromagen test) Procedure: Place sample of stain under coverslipProcedure: Place sample of stain under coverslip Heat while add pyradine in alkaline solutionHeat while add pyradine in alkaline solution Salmon colored crystals form if blood presentSalmon colored crystals form if blood present *Do you think labs do this test today?*Do you think labs do this test today? * Why/why not?* Why/why not?

44. Species Origin Diffusion reaction testDiffusion reaction test OuchterlonyOuchterlony Antibody-antigen reactionAntibody-antigen reaction Human blood & human antiserumHuman blood & human antiserum Process:Process:

45. Semen Gelatinous mixture of cells, amino acids, sugars, salts, ions, and other materialsGelatinous mixture of cells, amino acids, sugars, salts, ions, and other materials Spermatozoa = sperm cellsSpermatozoa = sperm cells Volume of ejaculation varies from 2 – 6 mL and contains 100-150 million spermatozoaVolume of ejaculation varies from 2 – 6 mL and contains 100-150 million spermatozoa

46. Presumptive Tests for Semen Acid phosphatase AP in semen is an enzymeAcid phosphatase AP in semen is an enzyme Brentamine Fast Blue B (known carcinogen)Brentamine Fast Blue B (known carcinogen) Prepare paper or swabPrepare paper or swab Apply chemical- if turns purple (in 2 minutes), positive for semenApply chemical- if turns purple (in 2 minutes), positive for semen If sample is aged, neg resultIf sample is aged, neg result Positive result may occurPositive result may occur

47. Presumptive tests for semen Seen with ALS (alternate light source and amber goggles)Seen with ALS (alternate light source and amber goggles) Covers large areasCovers large areas Disadvantage- some other fluids may be highlighted (urine and saliva)Disadvantage- some other fluids may be highlighted (urine and saliva) Just because no light- can’t assume no semenJust because no light- can’t assume no semen

48. Confirmatory tests for semen Christmas tree stainChristmas tree stain Tip of sperm’s head turns pink, bottom of head dark red, middle portion blue, tail yellowish-greenTip of sperm’s head turns pink, bottom of head dark red, middle portion blue, tail yellowish-green Skin cells stain green blueSkin cells stain green blue

49. Confirmatory tests for semen Time Since Intercourse (TSI)Time Since Intercourse (TSI) Motile sperm can survive in vagina for about 3 hours (1-8 hrs)Motile sperm can survive in vagina for about 3 hours (1-8 hrs) And up to several days in cervixAnd up to several days in cervix Intact sperm survive in vagina up to 26 hoursIntact sperm survive in vagina up to 26 hours Heads- up to 3 daysHeads- up to 3 days

50. Saliva Where can saliva be found?Where can saliva be found? Bite marksBite marks Licked adhesivesLicked adhesives Eating and drinking surfacesEating and drinking surfaces Spitting (expectoration)Spitting (expectoration) DETECTION CAN BE TRICKYDETECTION CAN BE TRICKY Amylase!Amylase! Found in high quantities in saliva, but also in other body fluidsFound in high quantities in saliva, but also in other body fluids If presumptive positive for saliva, sent for DNA analysisIf presumptive positive for saliva, sent for DNA analysis

51. Urine What is urine?What is urine? Excreted fluid and waste products by kidneysExcreted fluid and waste products by kidneys Detect presence of ureaDetect presence of urea Or creatineOr creatine

52. Checkpoint Why would TSI be advantageous to investigate?Why would TSI be advantageous to investigate? What is the most common presumptive test for semen?What is the most common presumptive test for semen? Brentamine fast blue BBrentamine fast blue B What is the difference between a presumptive and confirmatory test?What is the difference between a presumptive and confirmatory test? List 3 presumptive tests for blood.List 3 presumptive tests for blood.

53. Additional Blood Proteins TypePercentFraction MM30%30/100 MN48%48/100 NN22%22/100 Other blood proteins exist…Other blood proteins exist… Inherited from our parentsInherited from our parents The percentages of these other proteins are:The percentages of these other proteins are:

54. Blood-Type Probability Knowing the frequency of different genes in a population..Knowing the frequency of different genes in a population.. Determine probability/chance that a blood type will appearDetermine probability/chance that a blood type will appear

55. Example Problem What percentage of the population would have A+ blood?What percentage of the population would have A+ blood? Type A blood = 42% of population & Rh+ = 85% of populationType A blood = 42% of population & Rh+ = 85% of population 1. Convert % to decimals1. Convert % to decimals Type A = 0.42 of the populationType A = 0.42 of the population Rh+ =.85 of populationRh+ =.85 of population 2. Multiple decimals2. Multiple decimals 0.42 x 0.85 = 0.3570.42 x 0.85 = 0.357 3. Multiply by 100 to convert to %3. Multiply by 100 to convert to % 0.357 x 100% = 35.7%0.357 x 100% = 35.7% …about 36 out of every 100 people would have Type A+…about 36 out of every 100 people would have Type A+

56. Why is Blood Type probability Helpful? We can limit the size of our suspect population and help identify a suspectWe can limit the size of our suspect population and help identify a suspect If blood is found at crime scene with the following proteins: Type A, N, Hp-1, Rh-, PGM-2If blood is found at crime scene with the following proteins: Type A, N, Hp-1, Rh-, PGM-2 Probability = 1 in every 8600 people!Probability = 1 in every 8600 people! The more blood type proteins identified, uniqueness increases - number of people with same combo as suspects decreasesThe more blood type proteins identified, uniqueness increases - number of people with same combo as suspects decreases

57. Blood Evidence Blood samples – Can be analyzed to determine blood type and DNA, which can be matched to possible suspects. Blood samples – Can be analyzed to determine blood type and DNA, which can be matched to possible suspects. Blood droplets – Can be analyzed to give clues to the location of a crime, movement of a victim, and type of weapon. Blood droplets – Can be analyzed to give clues to the location of a crime, movement of a victim, and type of weapon. Blood spatter – Can be analyzed to determine patterns that give investigators clues to how a crime might have happened. Blood spatter – Can be analyzed to determine patterns that give investigators clues to how a crime might have happened.

58. Is it Human Blood? The method used is similar to that we use when our bodies recognize and attack invading substancesThe method used is similar to that we use when our bodies recognize and attack invading substances What two structures are involved in our immune response?What two structures are involved in our immune response? Antibodies and antigensAntibodies and antigens What does an antibody look like? Draw it.What does an antibody look like? Draw it.

59. ImmunoAssay Also known as immunoprecipitation assayAlso known as immunoprecipitation assay What two words do you see in immunoprecipitation?What two words do you see in immunoprecipitation? Immune and precipitationImmune and precipitation Precipitin results when antibodies and antigens interactPrecipitin results when antibodies and antigens interact

60. Process for immunoassay Antiserum is developedAntiserum is developed Recall how antiserum is produced for human blood & share this with your neighborRecall how antiserum is produced for human blood & share this with your neighbor Imagine you are a scientist needed to confirm the presence of bear blood. How would you go about this detection?Imagine you are a scientist needed to confirm the presence of bear blood. How would you go about this detection?

61. Precipitin An antibody which can precipitate out of a solution upon antigen bindingAn antibody which can precipitate out of a solution upon antigen binding We see a foggy/murky/cloudy areaWe see a foggy/murky/cloudy area The precipitin forms where matching antibodies and antigens meetThe precipitin forms where matching antibodies and antigens meet

62. Precipitin Test Steps of the Precipitin Test 1.Once the antiserum or antibodies are created 2.They are applied to the unknown blood on a solid substance (agar plate) 3.A preciptin line is formed between the antibodies and antigens that match 1.A preciptin line is a region on the agar plate that becomes cloudy or murky where the antigen and antibody meet and interact

63. Precipitin

64. Checkpoint What characteristic of a antibody allows it to identify a particular antigen?What characteristic of a antibody allows it to identify a particular antigen? Specificity!Specificity! Are immunoassay tests such as the immunoprecipitation assay an example of presumptive or confirmatory testing? Why?Are immunoassay tests such as the immunoprecipitation assay an example of presumptive or confirmatory testing? Why? Confirmatory… only will identify one specific antigenConfirmatory… only will identify one specific antigen

65. Serology A broad scope of laboratory tests that use specific antigen and serum antibody reactionsA broad scope of laboratory tests that use specific antigen and serum antibody reactions Serum: the yellowish liquid that separates from the blood when a clot is formedSerum: the yellowish liquid that separates from the blood when a clot is formed

66. Blood typing OBJECTIVE: to determine the effect of the angle of impact and height on a blood dropOBJECTIVE: to determine the effect of the angle of impact and height on a blood drop DO NOW:DO NOW: Make 3 observations about the first set of blood drops;Make 3 observations about the first set of blood drops; Make 3 observations about the second set of blood dropsMake 3 observations about the second set of blood drops

67. Vocabulary Blood SPATTER: term that describes a stain that results from blood hitting a target Angle of Impact: angle created between the path the blood travels and the ground surface or floor

68. Summary Blood consists of cellular components and plasma containing dissolved ions, proteins and other moleculesBlood consists of cellular components and plasma containing dissolved ions, proteins and other molecules Blood types result from the presence of proteins on the surface of red blood cells and vary among individual humans. This variation can be used to exclude blood samples from belonging to a group of individualsBlood types result from the presence of proteins on the surface of red blood cells and vary among individual humans. This variation can be used to exclude blood samples from belonging to a group of individuals Other proteins in the blood, such as enzymes, also show variation and can be used as class evidenceOther proteins in the blood, such as enzymes, also show variation and can be used as class evidence Combinations of specific blood and enzyme types can narrow the possible source of a blood sample to a fairly small group of individualsCombinations of specific blood and enzyme types can narrow the possible source of a blood sample to a fairly small group of individuals

69. Summary Blood splatter analysis can be used to recreate a crime sceneBlood splatter analysis can be used to recreate a crime scene The angle of impact is calculated as the arc sine = width / lengthThe angle of impact is calculated as the arc sine = width / length The characteristics of blood drops on surfaces can show how the blood was deposited, at what rate the blood was moving, the location of the origin or source of blood, and the direction on which the blood was moving when it struck the surfaceThe characteristics of blood drops on surfaces can show how the blood was deposited, at what rate the blood was moving, the location of the origin or source of blood, and the direction on which the blood was moving when it struck the surface Crime scene investigators used several tests to locate and identify blood at a crime scene, including visualization with Luminol and identification with the Kastel-Meyer testCrime scene investigators used several tests to locate and identify blood at a crime scene, including visualization with Luminol and identification with the Kastel-Meyer test