1. Forensic Biology Screening Workshop
Blood

2. What Is Blood?
Slightly alkaline fluid made up of water, cells, enzymes, proteins, glucose, hormones, organic and inorganic substances
Circulates throughout body
Supplies nutrients and oxygen to body
Removes waste
Also carries hormones, vitamins, antibodies

3. Blood Cells Blood cells are made in the bone marrow Hemopoiesis
Starts as a “stem cell” (blasts)
Some immature blood cells stay in the bone marrow to mature
Others travel to other parts of the body to mature (lymph nodes, spleen, liver)

4. Blood Cells
Cells mature and differentiate into several classes of cells:
Red blood cells
White blood cells
Platelets

5. Red Blood Cells (Erythrocytes)
Have no nucleus
Not useful for DNA analysis
6-8 µm in size
~45% total volume of blood
Most abundant cell in the blood

6. Red Blood Cells
Disk shaped cells which make up 99% of the cells in the blood
Principal carriers of the red colored hemoglobin molecules.
Hemoglobin is an iron containing protein and binds about 97% of all oxygen in the body.

7. Red Blood Cells

8. Hemoglobin Contains 4 subunits Globular proteins with heme
Heme groups contain iron which binds O2
Arterial Blood: bright red when hemoglobin carries O2
Venous Blood: dark red (veins appears blue in color due to optical effects of light when hemoglobin lacks O2)
Heme is a non-protein

9. Hemoglobin

10. Heme With Iron Iron is held in this heterocyclic porphyrin ring.
The iron is bonded equally to all 4 nitrogens. 2 additional bonds are perpendicular to the plane – one is connected to the protein and the other is available to bind oxygen.

11. White Blood Cells (WBC) (Leukocytes)
Produced in bone marrow
WBCs have a nucleus
Useful for DNA analysis
Vital source of defense against external organisms
White blood cells also clean up dead cells and tissue debris that would otherwise accumulate and lead to problems.

12. White Blood Cells Five classes of leukocytes: Neutrophil Eosinophil
Basophil
Monocyte
Lymphocyte

13. Red and White Blood Cells
image courtesy of the U.S. National Institutes of Health

14. Platelets
Irregularly-shaped, colorless bodies produced in the bone marrow
Their sticky surface lets them, along with other substances, form clots to stop bleeding
Only active when damage occurs to the circulatory system walls

15. Hemostasis

16. Plasma Liquid portion of blood
Composed of water, proteins, electrolytes
blood cells and platelets are suspended in plasma
Regulates osmotic pressure
The transport medium for:
Glucose
Lipids
Hormones
Oxygen (not as much as heme)
Clotting factors
Waste

17. Serum Clear liquid that is left after blood coagulates
Plasma without the clotting factors

18. Forensic Significance Of Blood
Hemoglobin (RBC)
Peroxidase-like activity can cleave H2O2
Blood Group Antigen (RBC)
Bound to RBC membrane (ABO groups)
DNA (WBC)
Found in cells with nucleus
Proteins (PLASMA)
Serum used in species testing

19. Forensic Testing Presumptive tests Indicates a substance is present
Not specific
Confirmatory tests
Confirm a substance is present
Specific

20. Presumptive Tests Kastle-Meyer (Phenolphthalein)
Leucomalachite Green (LMG)
Hemastix
Luminol
Other tests:
Tetramethylbenzidine (TMB)
Benzidine
Ortho-tolidine
Ortho-toluidine

21. Presumptive Tests CATALYTIC TESTS
Based on the fact that hemoglobin (and some of its derivatives) exhibit a peroxidase activity
Tests based on this property are generally named after the compound undergoing oxidation (benzidine, TMB, etc.) or the discoverer (Kastle-Meyer, etc.)
Oxidant (hydrogen peroxide) oxidizes a colorless reagent to a colored reagent
Heme catalyzes this oxidation by cleaving an oxygen from hydrogen peroxide (H2O2)

22. Kastle-Meyer (KM) Reaction

23. Kastle-Meyer Test HOW TO MAKE:
Phenolphthalein + Potassium Hydroxide + Zinc
Reflux – boil / condense
Reduces phenolphthalein to phenolphthalin
Oxygen removed and combined with OH from potassium hydroxide – boils off as water
Becomes a colorless solution
Working solution
Phenolphthalin + ethanol + zinc pellets
Requires a lot of time to prep ~2-3 hours for refluxing

24. Kastle-Meyer Test HOW TO STORE: Amber bottle Light affects stability
Zinc pellets
Binds free oxygen, prevents oxidation
Room temp (working solution)
Refrigerate (stock solution)

25. Kastle-Meyer Test HOW TO PERFORM:
Place a small cutting, swabbing, or extract of the suspected bloodstain on filter paper
- OR -
Swab the stain using a slightly moistened swab

26. Kastle-Meyer Test 3 STEP TEST STEP 1:
Add 2-3 drops of ethanol to the stain or swabbing
Note: This will increase sensitivity by cleaning the area around the hemoglobin, better exposing the heme

27. Kastle-Meyer Test STEP 2: Add 2 drops of reagent Wait for ~5 seconds
Note: This step aids in ruling out false positives due to the presence of chemical oxidants such as rust.

28. Kastle-Meyer Test Add 2-3 drops of 3% H2O2
STEP 3:
Add 2-3 drops of 3% H2O2
If immediate color change to PINK – the test is POSITIVE for the possible presence of blood
If no color change – blood is not present or is in too limited quantity for the test to detect.
Note: swab will eventually turn pink (even if negative) over time due to nature of oxidation reactions.
A color change should occur in 10 seconds or less

29. Kastle-Meyer Test LIMITATIONS Sensitivity
1 in 105 to 1 in 106 on dried stains
Specificity
Can weed out false positives between steps 2 and 3
Chemical oxidants, vegetable peroxidases
Will not detect differences in animal or human blood
Stability
Relatively stable if the reagents are stored separately and refrigerated.
Km one of the most widely used tests due to its safety factor & sensitivity
Sensitivity differs in articles. I saw ~1:1000 in my training samples. Would be a good exercise to do in your own lab.
Note: With all catalytic tests, high levels of bleach can cause false positives

30. Kastle-Meyer Video

31. Leucomalachite Green (LMG) Reaction

32. Leucomalachite Green HOW TO MAKE:
Malachite green + acetic acid + water + Zinc
Reflux – boil / condense
Becomes a colorless solution
Working solution
LMG reagent + zinc pellets
Similar to KM prep – requires time

33. Leucomalachite Green HOW TO STORE: Amber bottle Zinc pellets
Relatively stable at room temp

34. Leucomalachite Green HOW TO PERFORM:
Place a small cutting, swabbing, or extract of the suspected bloodstain on filter paper
- OR -
Swab the stain using a slightly moistened swab

35. Leucomalachite Green HOW TO PERFORM: STEP 1:
Add 1-2 drops of LMG reagent
Note color change, if there is a color change, the test is considered inconclusive

36. Leucomalachite Green HOW TO PERFORM: STEP 2: Add 1-2 drops of H2O2
Note results
If color change to deep green-blue, the test is positive for the possible presence of blood
If no color change the test is negative

37. Leucomalachite Green LIMITATIONS: Sensitivity ~1:1000 Specificity
Chemical oxidants, vegetable peroxidases
Will not detect differences in animal or human blood
Stability
Similar to KM
Less specific then KM b/c doesn’t rule out vegetable peroxidases as well.

38. Leucomalachite Green Video

39. Hemastix Reagent strips Bottle of 50 reagent strips Store at room temp
Test is based on the peroxidase activity of hemoglobin
Originally designed to detect blood in urine

40. Hemastix
Reagent on hemastix is diisopropylbenzene dihydroperoxide and ,3’,5,5’-tetramethylbenzidine (TMB)
Color change ranges from orange to green
Possibly blue with higher concentrations of blood

41. Hemastix HOW TO PERFORM: STEP 1:
Slightly moisten the pad on the tip of the strip with water
STEP 2:
Rub the damp pad on the stain in question
Note color change within 60 seconds and compare to the chart on the bottle
more green indicates more hemoglobin

42. Hemastix LIMITATIONS Sensitivity = 0.015-0.062 mg/dL free hemoglobin
Specificity
Chemical oxidants, vegetable peroxidases
Will not detect differences in animal or human blood
Stability
Stable for ~ 1 year – date stamped expiration date on bottle

43. Hemastix Video

44. Luminol

45. Luminol
HOW IT WORKS:
The iron in hemoglobin acts as a catalyst to cause a reaction between the luminol and H2O2.
Luminol loses nitrogen and hydrogen and gains oxygen.
This results in 3-aminopthalate which is energized and emits light

46. Luminol HOW TO MAKE: Reagents needed: Luminol (3-aminophthalhydrazide)
Sodium Perborate
Distilled water
Sodium Carbonate

47. Luminol HOW TO STORE: Spray bottle works best for testing
Make each time you use it (daily)

48. Luminol HOW TO PERFORM: STEP 1:
Spray the luminol directly onto the stain in question
Note: This test needs to be done in the dark to see the luminescence reaction which can last for approximately 15 seconds
If the stain emits a light then the test result is POSITIVE for the possible presence of blood
If there is no reaction the result is NEGATIVE

49. Luminol LIMITATIONS: Sensitivity
10-6 to 10-8 – most sensitive presumptive test
Specificity
Many false positives – bleach, metals, chemical oxidants, vegetable peroxidases
Will not detect differences in animal or human blood
Stability
Very unstable ~8 hour limit
Mostly used at crime scene
Can dilute out stain (possibly too much for DNA analysis)
Used more for blood spatter, crime scene reconstruction
If a stain is invisible to the naked eye and then you douse it with luminol, will dilute out any remaining stain. Not much hope for obtaining enough sample to get a DNA profile.

50. Tetramethylbenzidine (TMB)

51. Tetramethylbenzidine (TMB)
HOW TO MAKE:
3,3’, 5,5 ‘-tetramethylbenzidine (TMB) + glacial acetic acid
Easy to make compared to KM and LMG reagents

52. Tetramethylbenzidine (TMB)
HOW TO STORE:
Amber bottle
Light affects stability
Refrigerate between uses – only good for ~ 1 week.
Best to make fresh for each use - will decrease in sensitivity

53. Tetramethylbenzidine (TMB)
HOW TO PERFORM:
Place a small cutting, swabbing, or extract of the suspected bloodstain on filter paper
- OR -
Swab the stain using a slightly moistened swab

54. Tetramethylbenzidine (TMB)
HOW TO PERFORM:
STEP 1:
Add one drop of TMB solution
STEP 2:
Add one drop of 3% H2O2
Detect color change:
If the stain turns blueish green, the test result is POSITIVE for the possible presence of blood
NEGATIVE if no color change

55. Tetramethylbenzidine (TMB)
LIMITATIONS
Sensitivity
1:10,000 on dried stains
Specificity
Not as specific as KM test
False positives to vegetable peroxidases, bleach, potassium permanganate
Will not detect differences in animal or human blood
Possibly unsafe – labs beginning to eliminate use and use LMG or hemastix

56. Tetramethylbenzidine (TMB)
LIMITATIONS
Stability
Very unstable – 1 week maximum
Loses sensitivity by a factor of 10 after 1 day
Safety
Mutagen

57. Other Tests
Benzidine
Ortho tolidine (o-tolidine) - 3,3'-Dimethylbenzidine
Increased sensitivity, decreased specificity, and same stability when compared to Kastle-Meyer
Rarely used due to safety concerns- carcinogenic

58. Species Testing Definitions:
Antigen (Ag) – a soluble protein that causes the production of an antibody. Usually foreign to the body.
Antibody (Ab) – A “Y” shaped immunoglobulin produced by B-lymphocytes in the bone marrow.
Produced in response to an antigen.

60. Species Testing
Ag and Ab bind together to create a lattice network of molecules
This becomes insoluble and precipitates out of solution

61. Species Testing To make the antibodies: Host animal Goat, rabbit
Inject with human serum
Makes antibodies in response
Collect heart blood and test for Ab
Sample extracts are made from stained areas of interest, and from nearby unstained areas (substrate controls). 
Note that the use of unstained controls is a fundamental principle in forensic immunologic testing.

62. Tube Method
The original precipitin reaction was carried out by layering a solution of antibody on top of a solution of stain extract in a tube
This mixture was left for a period of time to allow the development of a precipitin band at the interface.
This is referred to as the tube method, and is still used in a few laboratories today

63. Ouchterlony
Method published in 1948 and uses radial diffusion of the antigen and antibody through agar gel.
How to perform:
Extract the stain in a microcentrifuge tube with saline. Extract should be straw colored.
Stain and controls samples are loaded in the outer wells and a drop of anti-human antiserum is loaded into the center well.  The process is repeated for antisera to other species, such as dog, cat, and cow; this may include the species from which the antiserum was obtained (e.g., rabbit).

64. Ouchterlony
The plates are left at room temperature or 37°C for a suitable period (which can range from a few hours to overnight) and the serum proteins and antibody molecules diffuse outward from the wells. 
A precipitin band is formed when the diffusing stain contains proteins that are recognized by IgG molecules in the diffusing antiserum. 
The precipitin band is sometimes clearly visible to the naked eye, but it is normal to stain the plates with amido black, Coomassie Blue, or other general protein stain, to enhance sensitivity and clarity.

65. Ouchterlony
If a white line occurs between the samples, then the test sample is positive for human antigen activity (or whatever species is being tested)
If no white line occurs then the test sample is negative for human antigen activity (or whatever species is being tested)

66. Ouchterlony Sensitivity 10-1 Specificity False positives: Detergents
Tannic Acid
False negatives:
Excess Ab or Ag – the lattice will not form, no precipitate

67. Discuss partial identity

68. Ouchterlony
Punch holes in the plate with 1 in the center and 6 around it. Add the Ab to the center well and the stain extract and controls to the outer wells. Should use antibodies from the animal used to create the anti-human antibodies to ensure there would be no cross reaction with human blood.

69. Ouchterlony Video

70. Cross-over Electrophoresis
A variant of the Ouchterlony test, which uses an electric field rather than diffusion to move the extract and antibody through the gel.
Cross-over electrophoresis for species identification is conducted using agar at a pH of 8.6. 
Stain extracts are loaded into wells arranged in a line at the cathode end of the plate and the antiserum is loaded into wells at the anode end. 

71. Cross-over Electrophoresis
During electrophoresis, the electric field drives the serum proteins towards the anode, but the IgG molecules, which are essentially neutral at this pH, are driven to the cathode by the process of electroendosmosis. 
The antigen-antibody precipitation occurs at the interface between the two rows of wells. 

72. Cross-over Electrophoresis
Electroendosmosis occurs because the supporting medium acquires a net negative charge.
If free, the negatively charged molecules would migrate to the anode, but this is not possible because the agar is immobilized on the plate. 
Instead, the effect is countered by positively charged water molecules migrating to the cathode.  The migrating water molecules carry any dissolved neutral molecules (such as IgG) with them.

73. ABAcard® Hematrace® Tests for human hemoglobin (Hb)
If human Hb is present – reacts with a mobile monoclonal anti-human HB antibody
Forms a mobile Ag-Ab complex
This migrates to the “T” zone

74. ABAcard® Hematrace®
In the “T” zone – polyclonal antihuman HB antibodies.
Forms Ab-Ag-Ab complex
Antibodies tagged with pink dye – upon aggregation at “T” zone – pink line
Control zone – has immobile anti-immunoglobulin which binds excess antihuman HB antibodies – form a pink line

75. ABAcard® Hematrace® To perform:
Extract cutting from stain in ~300 µl buffer
Leave 1-5 minutes
Add 150 µl of sample to the “S” well of the test card
Wait 10 minutes – read results
Pink line in “T” and “C” zones = POSITIVE
Pink line in only “C” zone = NEGATIVE

76. ABAcard® Hematrace® Limitations:
High Dose Hook Effect – can give a false negative
Occurs with excess hemoglobin which binds to the stationary antihuman HB Antibody in the “T” area
This prevents the mobile Ab-Ag complex from binding
Results in no pink line = NEGATIVE
FALSE NEGATIVE
Not a confirmatory test – gives positive result with ferret blood

77. ABAcard® Hematrace®

78. Hematrace Video

79. Seratec  HemDirect Hemoglobin Assay
Tests for human hemoglobin (Hb)
Similar to the ABAcard® Hematrace® test

80. Hematin Crystals Teichmann published method in 1853
Blood is treated with a solution of potassium bromide, potassium chloride and potassium iodide in glacial acetic acid, and is heated to react with hemoglobin.  The reaction first converts the hemoglobin to hemin, and then the halides react with the hemin to form characteristic brownish-yellow rhomboid crystals.
The test went through many modifications
Test did not always work even when the presence of blood was known

81. Hemochromagen Crystals
Many variations were used
Takayama described his method in 1912, and it became the preferred method. His name is commonly used to describe the reagents and procedure he devised and also the hemochromogen crystals obtained
Blood is treated with a saturated dextrose solution, sodium hydroxide, pyridine and water. Ferrous iron from hemoglobin reacts with pyridine to produce red feathery crystals of pyridine ferroprotoporphyrin

82. Takayama Crystals Red feathery appearance
image courtesy Steve Gilbert, M.F.S., PhD -- SUNY at Canton

83. Crystal Tests Crystals are unique to blood Not human specific
Not widely used anymore
Combo KM and ABA card
Time/money constraints
DNA testing is higher primate specific
Use of qPCR

84. Controls
Positive
Negative
Substrate

85. Positive Controls Used to determine if the tests are working properly
a quality control check
This is documented in the case file
Example: Known blood on cloth or a swab

86. Negative Controls
Used to determine if the reagents are contaminant free and working properly
a quality control check
This is documented in the case file
Example: unstained cloth or a swab

87. Substrate Controls
Used to determine if the substrate is interfering with the test
troubleshooting
This is documented in the case file
Example: unstained area adjacent to the questioned stain being tested