1. S
erology
By M. Hoffmeister & H. Montgomery

2. Identification & Character of Blood and Bloodstains
Forensic Science
Uses blood analysis to determine the individual
(source)
Usually deals with a variety of fluids (blood, saliva, semen, and urine) in stain form and are often degraded/deteriorated.
Environmental controls are essential ( uncontrolled heat and humidity can destroy a lot of biological information within a stain by enhancing degradation).
Serology
Chapter 11 & 12

3. Identification & Character of Blood and Bloodstains
Serology was very prominent in the 1950s to the 1980s, when DNA analysis became possible
It is still routinely done where DNA is analyzed.
Is done when DNA analysis is not feasible
Serology is being challenged by the reliability and the discrimination power of DNA analysis.
Important to understand preDNA serology f testimony in cases
Serology
Chapter 11 & 12

4. Protocol for the Analysis of Blood in Forensic Serology
Careful visualization of evidence to locate stains/
material visibly characteristic of blood.
Application of a suitable presumptive test.
Application of a confirmatory (specific and sensitive) test.
Determination of biological/species (animal or human) origin.
Characterization of blood using genetic markers or DNA.
Serology
Chapter 11 & 12

5. Identification of Blood
A visual observation coupled with positive chemical presumptive and confirmatory tests provide sound data to support the identification of blood.
Two types of tests are given because ----
NO SINGLE TEST IS ABSOLUTELY SPECIFIC
FOR BLOOD
Serology
Chapter 11 & 12

6. Serology Catalytic Color Tests Chapter 11 & 12
Catalytic Color Tests - employ chemical oxidation of a chromogenic (color causing) substance by an oxidizing agent catalyzed by the presence of hemoglobin (in red blood cells).
Two substances are needed for the color change:
Oxidizing agent (often hydrogen peroxide).
The heme group acts as a (peroxide – like) catalyst
Serology
Chapter 11 & 12

7. Catalytic Color Tests
Serology
Chapter 11 & 12

8. Serology Catalytic Color Tests Chapter 11 & 12
Common method of application:
Wet cotton swab with distilled water and sample the stain
Apply the reagents to the swab.
Note immediate observations – changes will take place quickly and may not appear the same after time has passed.
False positives can usually be attributed to:
Chemical oxidants
Plant materials (with peroxidase-like enzyme).
Animal materials that may contain traces of blood.
Serology
Chapter 11 & 12

9. Serology Catalytic Color Tests Chapter 11 & 12 Adler Test (Benzidine)
More extensively used than any other presumptive test for blood
Normally carried out in ethanol/acetic acid solution
Results in a characteristic blue color
Was deemed a carcinogen in 1974 and has been essentially discontinued for forensic use today
Serology
Chapter 11 & 12

10. Catalytic Color Tests
Adler Test (Benzidine)
Serology
Chapter 11 & 12

11. Serology Catalytic Color Tests Chapter 11 & 12
Kastle-Meyer Test (Phenolphthalein)
Commonly used today
Uses phenolphthalein (acid/base indicator)
Results in hot pink color
Phenolphthalein (alkaline solution) becomes oxidized to phenolphthalein (pink in alkaline environment)
Phenolphthalein over zinc in KOH solution
False positives do not usually produce the pink color
Serology
Chapter 11 & 12

12. Serology Catalytic Color Tests Chapter 11 & 12
Kastle-Meyer Test (Phenolphthalein)
Serology
Chapter 11 & 12

13. Serology Catalytic Color Tests Chapter 11 & 12
O-Tolidine (ortho-tolidine)
Derivative of benzidine
Conducted under acidic conditions
Creates a blue color (similar to benzidine)
Reported as carcinogenic in rats in 1974, leading to its replacement by TMB
Serology
Chapter 11 & 12

14. Serology Catalytic Color Tests Chapter 11 & 12
O-Tolidine (ortho-tolidine)
Serology
Chapter 11 & 12

15. Serology Catalytic Color Tests Chapter 11 & 12
Tetramethylbenzidine (TMB)
Derivative of benzidine
Used in an acidic medium (acetic acid)
Resulting color change from green to blue/green
Hemastix (field test for blood) uses TMB and only requires distilled water and the questioned sample (yellow to blue/green)
Swabs with cometic materials may produce false positives
Serology
Chapter 11 & 12

16. Serology Catalytic Color Tests Chapter 11 & 12
Tetramethylbenzidine (TMB)
Serology
Chapter 11 & 12

17. Serology Catalytic Color Tests Chapter 11 & 12
Tetramethylbenzidine (TMB)
Serology
Chapter 11 & 12

18. Serology Catalytic Color Tests Chapter 11 & 12
Leucomalachite Green (LMG)
Created in 1904 using the reduced form of the malachite green dye.
Produces a green color
Carried out in an acidic medium with hydrogen peroxide as the oxidizer
Serology
Chapter 11 & 12

19. Serology Catalytic Color Tests Chapter 11 & 12
Leucomalachite Green (LMG)
Created in 1904 using the reduced form of the malachite green dye.
Produces a green color
Carried out in an acidic medium with hydrogen peroxide as the oxidizer
Serology
Chapter 11 & 12

20. Serology Catalytic Color Tests Chapter 11 & 12
Tests using Chemiluminescence & Fluorescence
The observed results often enables one to determine the limits, shape and details of the original bloodstained area (and patterns if present)
By nature, they are potential sources of contamination of the blood
If the blood can be seen and collected, these probably should not be used
Luminol and Fluorescein are irritants but not carcinogens
Chemiluminescence is the process by which light is emitted as product of a chemical reaction
Fluorescence occurs when a chemical substance is exposed to a particular wavelength of light (usually a short wave like, UV light) and light energy is emitted at longer wavelengths
Serology
Chapter 11 & 12

21. Serology Catalytic Color Tests Chapter 11 & 12
Tests using Chemiluminescence
Serology
Chapter 11 & 12

22. Serology Catalytic Color Tests Chapter 11 & 12
Tests using Fluorescence
Serology
Chapter 11 & 12

23. Serology Catalytic Color Tests Chapter 11 & 12
Luminol (3-aminophthalhydrazide)
Luminesces after oxidation in acid or alkaline (aqueous) solution.
The catalytic activity of the heme group accelerates the oxidation of the luminol
Produces a blue-white to yellow-green light if blood is present
Outlines & details are often visible up to 30 second before additional spraying is required
Doesn’t affect these tests: presumptive, confirmatory, species origin, ABO tests, RFLP and PCR tests
Interferes with several enzyme and protein genetic marker systems
Sensitivity: detects dilutions up to 1 in 10,000,000
Results should be photographed b/c transient (temporary)
False positive with bleach (flash)
Serology
Chapter 11 & 12

24. Serology Catalytic Color Tests Chapter 11 & 12
Luminol (3-aminophthalhydrazide)
Serology
Chapter 11 & 12

25. Serology Catalytic Color Tests Chapter 11 & 12 Fluorescein
Prepared like phenolphthalein—reduced in an alkaline (basic) solution over zinc to fluorescin.
Fluorescin is applied to the sample and the heme group catalyzes the oxidation by hydrogen peroxide to make fluorescein.
When treated with UV light, it will fluoresce
Includes a commercial thickener which causes it to adhere to the surface (good for vertical surfaces).
Does not fluoresce with bleach
Shows no interference with STR testing of blood for DNA.
Serology
Chapter 11 & 12

26. Catalytic Color Tests
Fluorescein
Serology
Chapter 11 & 12

27. Confirmatory Test for Blood
(Crystal tests are often used)
Teichmann Test
First described in 1853
Heating dried blood in the presence of glacial acetic acid and a halide salt (usually chloride) to form a hematin derivative.
Serology
Chapter 11 & 12

28. Confirmatory Test for Blood
(Crystal tests are often used)
Teichmann Test
Serology
Chapter 11 & 12

29. Confirmatory Test for Blood
(Crystal tests are often used)
Takayama Test
First used in 1912
Involves heating dried blood in the presence of pyridine, a glucose (reducing agent), and sodium hydroxide (basic) solution to form a pyridine derivative.
Serology
Chapter 11 & 12

30. Confirmatory Test for Blood
(Crystal tests are often used)
Takayama Test
Serology
Chapter 11 & 12

31. Confirmatory Test for Blood
(Crystal tests are often used)
Teichmann Test
First described in 1853
Heating dried blood in the presence of glacial acetic acid and a halide salt (usually chloride) to form a hematin derivative.
Takayama Test
First used in 1912
Involves heating dried blood in the presence of pyridine, a glucose (reducing agent), and sodium hydroxide (basic) solution to form a pyridine derivative.
Serology
Chapter 11 & 12

32. Species Determination in Bloodstains
Microscopy uses a visual comparison of the blood cell morphology (shape/appearance)
Serology
Chapter 11 & 12

33. Species Determination in Bloodstains
Microscopy uses a visual comparison of the blood cell morphology (shape/appearance)
Protein analysis (3 different techniques are used.)
Immunoprecipitation reactions (most use)
If an animal is injected with a human serum protein, the animal’s immune system will recognize it as foreign and release produce antibodies against the antigen.
When the antibodies and antigen come in contact and usually a precipitation is formed (precipitin reaction)
Serology
Chapter 11 & 12

34. Species Determination in Bloodstains
Immunoprecipitation reactions
Serology
Chapter 11 & 12

35. Species Determination in Bloodstains
Protein analysis (continued)
Electrophoresis—a technique in which charged molecules (i.e. proteins) are caused to migrate in an electric field in a suitable support medium under controlled conditions of temp, pH, voltage, and time.
Support media: starch gels, agarose gels, polyacrylamide gels
Positively charged molecules migrate toward the cathode (- electrode)& negatively charged molecules migrate toward the anode (+ electrode).
Bands are visualized by a stain or chemical rxns.
In FS, it is usually done in a gel on a glass plate w/ samples (i.e. blood soaked thread or stain extracts) are placed in the gel.
Serology
Chapter 11 & 12

36. Species Determination in Bloodstains
Electrophoresis
Serology
Chapter 11 & 12

37. Species Determination in Bloodstains
Protein analysis (continued)
Isoelectric Focusing (IEF) - an electrophoretic method that takes advantage of the fact that at a certain pH, a protein in aqueous solution will exhibit a point of no net charge (isoelectric point - IEP).
The buffer in the gel controls the pH throughout the system
A pH gradient is set up in the gel w/ the low end at the anode.
When current is applied, the proteins migrate where they encounter their IEPs and form a band
The bands here can sometimes be more easily observed that with electrophoresis.
Serology
Chapter 11 & 12

38. Species Determination in Bloodstains
Isoelectric Focusing (IEF)
Serology
Chapter 11 & 12

39. Species Determination in Bloodstains
Serum Protein Analysis - serum proteins are a large collections of proteins in the serum
Ring Precipitin Test
2 liquids placed in a test tube: antiserum and extract of questioned bloodstain
As the anti-human serum comes in contact with human blood sample via diffusion, a small layer of a precipitate will form between these two liquids.
No reaction will occur if the blood is animal and used with anti-human serum.
Serology
Chapter 11 & 12

40. Species Determination in Bloodstains
Ring Precipitin Test
Serology
Chapter 11 & 12

41. Species Determination in Bloodstains
Serum Protein Analysis - serum proteins are a large collections of proteins in the serum
Ring Precipitin Test
2 liquids placed in a test tube: antiserum and extract of questioned bloodstain
As the anti-human serum comes in contact with human blood sample via diffusion, a small layer of a precipitate will form between these two liquids.
No reaction will occur if the blood is animal and used with anti-human serum.
Outerlony Double Diffusion Test
Carried out in a gel on a glass plate or petri dish
Wells are punched into agar, the antiserum is placed in the center well and the extracts are in the surrounding wells.
Immunoprecipitate lines form between the wells to show a positive response.
Serology
Chapter 11 & 12

42. Species Determination in Bloodstains
Outerlony Double Diffusion Test
Serology
Chapter 11 & 12

43. Species Determination in Bloodstains
Serology
Chapter 11 & 12

44. Species Determination in Bloodstains
Serum Protein Analysis (continued)
Crossed-Over Electrophoresis
Rows of opposing wells are cut in an agarose gel plate.
Once current is applied, the extract proteins (antigen) move toward the anode.
Antibodies move via electroendosmosis—cations and water of hydration move toward the cathode.
When the antibodies meet with the extract protein (antigen) precipitin bands form between the two rows of wells
Non-Serum Analysis includes using antihuman Hemoglobin
Hemoglobin is a protein in blood that is species-specific for immunological properties.
Serology
Chapter 11 & 12

45. Genetic Markers in Blood
Blood group - a group of antigens produced by allelic genes at a single locus and inherited independently of other genes.)
Serology
Chapter 11 & 12

46. Genetic Markers in Blood
Antigen-based Markers and Protein Markers
Antigen-Based Markers: Blood Groups
ABO system
Discovered by Landsteiner in 1900
Types (A, B, AB, O) refer to antigens on the surface of the RBC
Antibodies for the antigens (anti-A, anti-B) are present in the plasma.
Agglutination would occur between anti-A serum and types A and AB blood
Most individuals (80%) secrete their ABO characteristics in other body fluids (they are called secretors).
Serology
Chapter 11 & 12

47. Genetic Markers in Blood
Antigen-based Markers and Protein Markers
Antigen-Based Markers: Blood Groups
ABO system
Serology
Chapter 11 & 12

48. Genetic Markers in Blood
Blood Types
Serology
Chapter 11 & 12

49. Genetic Markers in Blood
Antigen-based Markers and Protein Markers
Antigen-Based Markers: Blood Groups
ABO system
The Lewis (Le) System
Le antigens are absorbed from the plasma onto the surface of RBCs.
Provide secretor status (whether the ABO type can be determined from bodily fluids).
Rhesus (Rh) system (+ or -)
Discovered by Landsteiner and Wiener (1940)
Antigens are on the surface of the RBC membrane
Not present in non-blood body fluids
Natural Rh antibodies are not common in serum
Serology
Chapter 11 & 12

50. Genetic Markers in Blood
Antigen-based Markers and Protein Markers
Protein Markers: Blood Groups
Hemoglobin (Hb)
Major protein in RBCs
Phenotypes can be identified w/ electrophoresis or IEF
Haptoglobin (Hp)
Enzyme Markers
uses electrophoresis and IEF (Isoelectric Focusing)
Phosphoglucomutase (PGM)
Most well known enzyme
Found in many tissues of plants, animals, & microorganisms
In humans, it exists in significant concentrations in blood, semen & in small amounts in vaginal secretions & cervical mucus.
Can place within 10 subtype population groups (most discriminatory of all enzyme systems used in Forensic Science)
Serology
Chapter 11 & 12

51. Genetic Markers in Blood
Haptoglobin
IEF – Isoelectric Focusing
Serology
Chapter 11 & 12

52. Serology Biological Fluids Chapter 11 & 12 Semen
Produced by males after puberty
Semi fluid mixture of cells amino acids, sugars, salts, organic & inorganic materials
Produced by seminal vesicles prostate gland and Cowper’s glands.
Ejaculation of human males: 2-6 ml with million sperm cells per ml
Genetic disorder, drug abuse, exposure to certain chemicals, etc. may decrease sperm count
Appears yellowish-white crust when dry
Serology
Chapter 11 & 12

53. Serology Biological Fluids Chapter 11 & 12 Sperm cells (spermatozoa)
First co-discovered by van leewenhoek in 1679 (he identified the first living cell)
Hartsoeker co-discovered sperm cells
Drew what he hoped to find (preformed human-“homunculus”) (1694)
Serology
Chapter 11 & 12

54. Hartsocker – drawing “homunculus”
Biological Fluids
Hartsocker – drawing “homunculus”
Serology
Chapter 11 & 12

55. Serology Biological Fluids Chapter 11 & 12 Sperm cells (spermatozoa)
First co-discovered by van leewenhoek in 1679 (he identified the first living cell)
Hartsoeker co-discovered sperm cells
Drew what he hoped to find (preformed human-“homunculus”) (1694)
About 55 m in length (90% is tail)
3 main parts of the sperm cell
Head
Contains the nucleus (DNA)
Acrosome: anterior portion that contains enzymes to penetrate the cell wall of the ovum during fertilization
Midpiece (attaches head & tail) contains the mitochondrion (provides energy for flagellated movement)
Tail—flagellum (means of locomotion)
Serology
Chapter 11 & 12

56. Biological Fluids
Sperm Cell Parts
Serology
Chapter 11 & 12

57. Serology Biological Fluids Chapter 11 & 12
Presumptive Testing for semen: Brentamine Fast Blue Test
The primary test for SAP (seminal acid phosphatase) activity
Produces a color reaction—purple color appears within 30 sec to 2 min.
Sensitive enough to produce a positive result with a dilution of 500 times
2 step procedure
Sterile water on swab/filter paper
Apply swab/filter paper to questioned stain
Reagent added to swab/filter paper
Color change noted
Several variations exist dependent on the sample
Serology
Chapter 11 & 12

58. Biological Fluids
Brentamine Fast Blue Test
Serology
Chapter 11 & 12

59. Serology Biological Fluids Chapter 11 & 12
Confirmatory Testing for semen
Microscopic Analysis
Christmas Tree Stain
Most common staining technique
Specifically developed for sperm cell identification: pink/red heads, blue midpiece, and green tail
Serology
Chapter 11 & 12

60. Serology Biological Fluids Chapter 11 & 12
Confirmatory Testing for semen
Microscopic Analysis
Christmas Tree Stain
Most common staining technique
Specifically developed for sperm cell identification: pink/red heads, blue midpiece, and green tail
Protein Analysis
p30 (PSA—prostate specific antigen)
the protein is found only in semen
uses sample and p30 antibodies—precipitin test
Serology
Chapter 11 & 12

61. Serology Biological Fluids Chapter 11 & 12
Confirmatory Testing for semen
Protein Analysis PSA
Serology
Chapter 11 & 12

62. Serology Biological Fluids Chapter 11 & 12 DNA sampling of sperm
Remember that a sperm cell only has 50% of the persons genes, you need about 80 spermatozoa to generate his complete DNA profile
PGM, Glycoxalase, Peptidase A are the identifiable enzymes in semen. (Allows one to narrow down rapist’s identity, but not as specific as blood.)
Serology
Chapter 11 & 12

63. Serology Biological Fluids Chapter 11 & 12 Collection of Rape Evidence
Has intercourse actually occurred? Semen inside of vagina, physical injuries (on victim)
Observe crime scene for signs of struggle
What needs to be collected?
Clothing - all (even shoes)
Bedding
Swabs - vaginal, anal, nasal and oral
Combings (head and pubic area) - take exemplars also
Blood - samples and exemplars
Saliva- samples and exemplars
Fingernail scrapings
If a suspect is taken into custody:
Take suspect’s clothing and examine for trace evidence (fiber/hair)
Examine suspect’s body for signs of a violent struggle (fingernail scratches, bite marks, etc.)
Hair and blood exemplars should be taken (legally)
Serology
Chapter 11 & 12

64. Serology Biological Fluids Chapter 11 & 12 Saliva
No specific test for saliva, but tests rely primarily on the presence of alpha-amylase (found in animals). [beta-amylase is found in plants]
2 DNA loci in humans found on chromosome 1:
AMY1 (codes for amylase found in saliva, breast milk, perspiration)
AMY2 codes for amylase found in pancreas, semen, and vaginal secretions
Limitation for testing is the presence of proteins from other body fluids may give false positives.
If DNA testing is important, it may be better not to consume the sample with body fluid testing (i.e. stamps, envelope flaps, bite marks).
Serology
Chapter 11 & 12

65. Biological Fluids
Saliva
Serology
Chapter 11 & 12

66. Biological Fluids – Saliva
Serology
Chapter 11 & 12

67. Serology Biological Fluids Chapter 11 & 12 Starch-Iodine Test
Earliest test devised in 1881
Iodine in starch (blue) + amylase  color changes (subsides)
Difficult for locator of stains
No way of quantifying amylase (just relative proportion)
Serology
Chapter 11 & 12

68. Biological Fluids
Starch-Iodine Test
Serology
Chapter 11 & 12

69. Serology Biological Fluids Chapter 11 & 12 Phadebas Reagent
Starch is linked to a dye molecule to form a coloration (light blue)
Press Test
Phadebas solution (dissolve tablet in dH2O) and spray on filter paper (allow to dry for storage)
Wet paper (if necessary) & outline paper on sample for reference
Press onto the sample with a weight and observe 40 min.
Test Tube
Variation of test involving liquid samples, centrifugation and analysis of supernatant liquid.
The coloration level is proportional to the amount of saliva, which can be measured by a spectrophotometer.
Serology
Chapter 11 & 12

70. Serology Biological Fluids Chapter 11 & 12
Phadebas Reagent – Press Test
Serology
Chapter 11 & 12

71. Biological Fluids – Blood/Sweat
Serology
Chapter 11 & 12

72. Serology Biological Fluids Chapter 11 & 12 Urine
Comprised of mostly water and salts
Cellular components are epithelial cells from the urinary tract (RBCs & WBCs)
Low cell concentrations in urine
Low rate of success in DNA testing (high volumes are better b/c they can be centrifuged)
Serology
Chapter 11 & 12

73. Serology Biological Fluids Chapter 11 & 12 Urine (continued)
Identification relies on identifying both urea and creatinine, which are present in high levels (found in other body fluids also).
Testing for urea:
Urea + enzyme (urease)ammonia + CO2
Ammonia is detected using an indicator such as Nessler’s reagent (mercuric iodide or potassium iodide) or DMAC (p-dimethylamino-cinnamaldehyde)
Testing for creatinine
Sample: + picric acid/toluene (or benzene)creatinine picrate
(creatinine) (colored product)
Serology
Chapter 11 & 12

74. Serology Biological Fluids Chapter 11 & 12 Feces
Solid waste products (undigested food residues, mucosal cells & bacteria)
Identifying characteristics:
Stains characterized by a green-brown color (due to bile pigments & products)
Distinct odor caused by the breakdown of amino acids by bacteria
Presence of undigested components of foodstuffs
Identified by the detection of urobilinogen (& urobilin products) - pigments (not present in herbivores)
Sample: + Alcoholic zinc acetate urobilin-zinc salt + urobilin
Urobilin-zinc salt fluoresces bright apple green w/ Long wave UV light.
DNA testing
Has been unsuccessful due to large amounts of bacteria & bile products
mtDNA has proven more successful
(urobilinogen)
Serology
Chapter 11 & 12

75. Serology Biological Fluids Chapter 11 & 12 Vomitus
No tests present for vomitus
Test for low pH from stomach acids
Amylase
Identification of foodstuffs
May help to determine time since death if the details of the last meal are known)
Serology
Chapter 11 & 12

76. Body Fluid Stains – Clothing
Serology
Chapter 11 & 12

77. Serology Biological Fluids Chapter 11 & 12 Vaginal Secretions
Usually identified based on glycogenated epithelial cells
Not unique to vaginal tract
These cells are absent in prepubescent and post menopausal females
Periodic acid-Schiff (PAS) reagent stains the glycogen in the cytoplasm a bright magenta
Testing may consume large amounts of DNA evidence
Serology
Chapter 11 & 12

78. Biological Fluids – Semen/Vaginal
Serology
Chapter 11 & 12