The document covers the fundamentals of forensic serology, emphasizing the significance of blood analysis in solving crimes through techniques such as blood type identification and blood spatter analysis. It outlines key learning outcomes for students, including understanding blood types, the importance of blood evidence in investigations, and methods for analyzing blood patterns. It also explains different tests for blood identification and characterization, as well as the principles and categories of blood spatter analysis.

1. ANALYSIS OF BLOOD
SBP4502 BASIC FORENSIC
Raj Cheema, PhD
Department of Biomedical Sciences, FMHS, UPM
manraj@upm.edu.my

2. ISLAM
&
FORENSIC
SCIENCE
Clear and defined guidance and framework with aims or goals
intended by Islamic law for the realization of benefit to mankind
Collection of proof that support each other till it form
a solid and defined conclusion/evidence

3. TABLE OF CONTENTS
The basics of blood
(pre-class activity)
Importance of blood
analysis in forensic
Blood spatter analysis Recap, case study and
reflection
(post-class activity)
01
03
02
04
BASICS
SPATTER
SEROLOGY
SUMMARY

4. At the end of the lecture, students would be able to;
I. Identify the fundamentals of basic serology and forensic serology.
II. Recognize the significance of blood analysis in forensic investigations.
III. Demonstrate an understanding of blood pattern analysis principles.
LEARNING OUTCOMES

5. https://www.newsweek.com/bryan-kohbergers-lawyer-fishing-info-about-potential-jurors-1884588

7.  Case #48311
 Discovery of an apparent homicide
 4 deceased, 1-knive, multiple blood stains discovered
 From the crime scene sketch, determine where the incident occurred? Height? Force?
And number of injuries?
REAL-CASE DEPICTION

8. 01
Comprehend the basics of forensic serology (Anatomy and Physiology)
BASICS (LO 1)

9. Serology (Blood Basics)
 What makes up our blood?
 Erythrocytes (RBC)
 Leucocytes (WBC)
 Thrombocytes (Platelets)
 Plasma
INTRODUCTION

10. Serology (Blood Facts)
 The average adult has about FIVE
liters of blood (7-8% of BW)
 There’s one billion red blood cells
in two to three drops of blood
 For every 600 red blood cells, there
are about 40 platelets and one white
cell
 Functions – O2, CO2, fights against
infection and helps heal wounds
INTRODUCTION

11. Serology (Blood Types)- Mixed
agglutination test
 There are 3 alleles for blood type: A, B, &
O.
 Since we have 2 genes, there are 6
possible combinations.
INTRODUCTION
Blood Types
AA or AO = Type A
BB or BO = Type B
OO = Type O
AB = Type AB

12. PRE-CLASS DATA
PutraBlast pre-class viewing quiz

13. PRE-CLASS DATA
Question
3
Question
6

14. 02
Understand the importance of blood analysis in
forensic
SEROLOGY (LO 1 & 2)

15. Importance of blood analysis in forensic science
 Blood is the most common and possibly most important evidence in the solving of certain types of
crimes.
 Nothing has been found to provide a better substitute for blood analysis. Its presence has the keen
ability to link a suspect and a victim, or a suspect to a location.
Blood evidence
 Blood samples – Can be analysed to determine blood type and DNA, which can be matched to
possible suspects.
 Blood droplets – Can be analysed to give clues to the location of a crime, movement of a victim, and
type of weapon.
 Blood spatter – Can be analysed to determine patterns that give investigators clues to how a crime
might have happened.
IMPORTANCE

16. History
 Dr. Eduard Piotrowski, assistant at the institute of Forensic Medicine at Poland, published first
paper on Blood Spattering in 1895.
 It was followed by French scientist Dr. Victor Balthazard in 1939.
IMPORTANCE

17. History
 Most profound work was done by
Professor Dr. Herbert Leon
MacDonell who had three
publications and several books
written on Blood Spattering from
1971 to 1982.
IMPORTANCE

18. The serology section of a forensic laboratory may deal with any or all the following:
 Blood typing/grouping
 Characterization of unknown blood
 Stain patterns for crime reconstruction
 DNA techniques used for identification
 Etc.
Bloodstain identification is commonly achieved by one of five methods of analysis:
i. Microscopic examination
ii. Chemical methods
iii. Spectrophoto-metric analysis
iv. Immunological methods
v. DNA analysis
FORENSIC SEROLOGY

19. Is it blood?
Is it human blood?
Can it be associated with an individual?
Question 1:
Question 3:
Question 2:
FORENSIC SEROLOGY
Forensic characterization of bloodstains.
Three questions that must be answered by the forensic investigator:

20. Is it blood?
Question 1:
FORENSIC SEROLOGY
Forensic characterization of bloodstains.
Three questions that must be answered by the forensic investigator:
Presumptive/Screening
• Benzidine Test
• Kastle-Meyer Test
• Hemastix (o-tolidine, TMB)
• Luminol Test
• Others (Guaiacum test, Aloin test,
Eosin, Rhodamine, etc.)
Confirmatory
• Takayama Test
• Teichmann Test
• Wagenaar Test
• RSID-Blood
• Others (microscopic, spectroscopic
examination, electrophoresis,
chromatography, RNA based assay,
etc.

21. Is it human blood?
Question 2:
FORENSIC SEROLOGY
Forensic characterization of bloodstains.
Three questions that must be answered by the forensic investigator:
 Precipitin Test
 Oakley-Fulthorpe test (Double diffusion test)
 Rapid immunoassay/ rapid immunochromatographic tests/lateral flow assays
 DNA analysis (Polymerase Chain Reaction (PCR) and Short Tandem Repeat (STR)
analysis)

22. Can it be associated with an individual?
Question 3:
FORENSIC SEROLOGY
Forensic characterization of bloodstains.
Three questions that must be answered by the forensic investigator:
 Blood typing reports / Medical Reports
 Police FIR/reports
 Etc.

23. Is it blood?
Question 1:
FORENSIC SEROLOGY
Recap:
Presumptive
• Benzidine Test
• Kastle-Meyer Test
• Hemastix (o-tolidine, TMB)
• Luminol Test
• Others (Guaiacum test, Aloin test,
Eosin, Rhodamine, etc.)
Confirmatory
• Takayama Test
• Teichmann Test
• Wagenaar Test
• RSID-Blood
• Others (microscopic, spectroscopic
examination, electrophoresis,
chromatography, RNA based assay,
etc.)

24. Forensic characterization of bloodstains. (Presumptive Tests)
In general, there are three broad categories on which the presumptive tests are divided to
justify whether the given stain is blood or not?
 Colour Test (visible color product) - action of the peroxidase enzyme in RBCs.
 Chemiluminescent Test - result in the release of light as a result of the catalytic
properties of blood.
 Fluorescent/luminescent Test - result in the release of light from a substance that has
absorbed light/energy previously.
FORENSIC SEROLOGY

25. Forensic characterization of bloodstains. (Presumptive Colour Tests)
Benzidine Test/Adler Test
FORENSIC SEROLOGY

26. Forensic characterization of bloodstains. (Presumptive Colour Tests)
Kastle-Meyer (Phenolphthalein) Test
FORENSIC SEROLOGY

27. FORENSIC SEROLOGY
Kastle-Meyer
(Phenolphthalein)
Test

28. Forensic characterization of bloodstains. (Presumptive Colour Tests)
Hemastix (o-tolidine, TMB)
 Change ranges from orange to green to possibly blue with higher concentrations
of blood
 Reagent on Hemastix® is diisopropylbenzene dihydroperoxide and 3, 3’, 5,
5’‐tetramethylbenzidine (TMB)
FORENSIC SEROLOGY

29. Forensic characterization of bloodstains. (Presumptive Colour Tests)
Leucomalachite green (LMG)
 Haemoglobin catalyses the reaction between LMG and hydrogen peroxide,
converting the colourless LMG into malachite green.
FORENSIC SEROLOGY

30. Forensic characterization of bloodstains. (Presumptive Chemiluminescent Tests)
Luminol/ Bluestar
 Luminol reacts with blood and hydrogen peroxide, producing blue-white to yellowish-
green light under very low light conditions (usually dark).
FORENSIC SEROLOGY

31. Forensic characterization of bloodstains. (Presumptive Chemiluminescent Tests)
Luminol/ Bluestar
 The possibility exists, however, that blood may be present in amounts too little to see
with the unaided eye, or that the blood at the scene had been “cleaned up” prior to
arrival of the crime scene team.
 The light, or luminescence, emitted in the luminol reaction results when an oxidising
agent, such as blood, catalyses the oxidation of luminol by hydrogen peroxide in a
basic solution.
 The reaction is not specific to blood.
 A follow up presumptive test, such as phenolphthalein, is typically run on potential
samples prior to collection.
FORENSIC SEROLOGY

33. A real case solved with
Luminol
https://youtu.be/k7czoo
qXsM4?feature=shared

34. Forensic characterization of bloodstains. (Presumptive Fluorescent Tests)
Fluorescein (Hemascien)
 Reacts with red blood cells to glow
 Needs an alternative light source to visualize (exposed/excited to light at the 420nm
to 485nm wavelengths)
FORENSIC SEROLOGY

35. Note:
How to choose the most
most suitable presumptive
presumptive test?
You need to acknowledge
acknowledge sensitivity,
sensitivity, false positive,
positive,
advantages and
disadvantages
https://forensicreader.com/presumptive-test-for-blood/

36. Forensic characterization of bloodstains. (Confirmatory Tests)
 To clearly depict whether the blood sample is blood or not, a special type of test is
needed to perform called confirmatory test. These special tests are highly specific to
blood i.e. nearly no false-positive result.
 Confirmatory blood tests depends majorly on the detection of Hb molecules in RBCs.
And in general scenarios, it is always done after the presumptive test for blood was
performed.
FORENSIC SEROLOGY

37. Forensic characterization of bloodstains. (Confirmatory Tests)
There are basically seven major types of confirmatory tests that can be used for the
confirmation of whether the possible bloodstain is actually blood or not? These are:
1. Microscopic Examination
 Intact RBCs
 WBCs Examination (Leishman stain)
2. Microcrystal Assay
 Teichmann (Hematin)
 Takayama Crystal Test
 Wagenaar Test
3. Spectroscopic Examination
4. Electrophoresis Method
5. Immunoelectrophoresis
6. RNA Based Assays
7. Chromatography Method
FORENSIC SEROLOGY
Good read: https://forensicreader.com/confirmatory-test-for-blood/

38. Forensic characterization of bloodstains. (Confirmatory Tests)
Microcrystal Assay
 A chemical test that results in the distinctive morphological development of
microscopic crystals as a sign of blood confirmation is called the microcrystal assay
test. These microscopic heme crystals can easily be seen from a standard microscope.
FORENSIC SEROLOGY
Good read: https://forensicreader.com/confirmatory-test-for-blood/

39. Forensic characterization of bloodstains. (Confirmatory Tests)
RSID-Blood
 Rapid stain identification-blood (RSID-blood) a lateral flow immuno-chromatographic
strip test format which allows the identification of human blood by detection
of glycophorin A, a red blood cell membrane antigen, using two anti-human
glycophorin A (GPA) monoclonal antibodies.
FORENSIC SEROLOGY
Good read: https://forensicreader.com/confirmatory-test-for-blood/

40. Is it human blood?
Question 2:
FORENSIC SEROLOGY
Forensic characterization of bloodstains.
Three questions that must be answered by the forensic investigator:
 Precipitin Test
 Oakley-Fulthorpe test (double diffusion test)
 Rapid immunoassay/ rapid immunochromatographic tests/lateral flow assays
 DNA analysis (Polymerase Chain Reaction (PCR) and Short Tandem Repeat (STR)
analysis)

41. FORENSIC SEROLOGY
 Precipitin Test (Ab-Ag Reaction)

42. FORENSIC SEROLOGY
Oakley-Fulthorpe test (Double Diffusion Test)
 Ab is incorporated in a gel, above which a column of
plain agar is placed
 The Ag is layered on top of the plain agar
 The Ag and Ab moved towards the plain agar through
the intervening column of plain agar
 This will result in the formation of precipitate when they
meet at optimum proportion.

43. Can it be associated with an individual?
Question 3:
FORENSIC SEROLOGY
Forensic characterization of bloodstains.
Three questions that must be answered by the forensic investigator:
 Blood typing reports
 Police FIR/reports
 Does the bloodstain appear to be related to the primary crime scene, or could it be a
result of secondary transfer?
 Are there any additional bloodstains or related evidence nearby that require
examination?
 Would further laboratory analysis, such as DNA profiling or serological testing, be
beneficial in aiding the investigation?
 WILL THE BLOODSTAIN TELL A STORY?

44. Take 5

45. LET’S PLAY

46. 03
Grasp the blood spatter analysis concept
SPATTER (LO 3)

48. BLOOD PATTERN ANALYSIS

49.  Bloodstain spatter/pattern analysis is the examination of the shapes, location &
distribution patterns of bloodstains, in order to provide an interpretation of the physical
events which give rise to their origin.
 Information that are likely to be discovered;
 The direction a given droplet was traveling at the time of impact.
 The angle of impact.
 The probable distance from the target from which the droplet originated.
 The nature of the force involved.
 The nature of object used.
 The relative position of the suspect, victim and other related objects.
 The approximate numbers of blows struck during an incident.
 Sequencing of multiple events associated with an incident
BLOOD PATTERN ANALYSIS

50. Terminologies:
BLOOD PATTERN ANALYSIS

51. Blood Spatter/Pattern Analysis will delve into;
I. Categories of Bloodstain Patterns
II. Directionality of Bloodstain
III. Impact Angle Determination
IV. Target Surface and Stain Shape
V. Point of Convergence
VI. Point of Origin
BLOOD PATTERN ANALYSIS

52. I. Categories of Bloodstain Patterns
a) Passive Bloodstains b) Transfer Bloodstains
BLOOD PATTERN ANALYSIS
c) Projected Bloodstains

53. a) Passive Bloodstains
 Are drops created or formed by the force of gravity acting alone.
 It can be further sub-divided to include:
 Drops
 Drip patterns
 Pools
 Clots
BLOOD PATTERN ANALYSIS

54. b) Transfer Bloodstains
 It is created when a wet, bloody surface comes in contact with a secondary surface.
 It is further sub-divided as:
 Contact/Transfer - an image is recognizable and may be identifiable with a particular
object.
 Swipe/Smear - wet blood is transferred to a surface that did not have blood on it.
 Wipe/Smudge - a non-blood-bearing object moves through a wet bloodstain, altering
the appearance of the original stain.
BLOOD PATTERN ANALYSIS
Contact/Transfer Swipe/Smear Wipe/Smudge

55. TEST YOURSELF
Contact/Transfer
Swipe/Smear
Wipe/Smudge

56. c) Projected Bloodstains
 These are created when an exposed blood source is
subjected to an action or force, greater than the force of
gravity.
 It can be divided into:
 Arterial Spurt - Bloodstain pattern(s) resulting from
blood exiting the body under pressure from a
breached artery
 Cast-off Stains - Blood released or thrown from a
blood-bearing object in motion
 Impact Spatter - Blood stain patterns created when a
blood source receives a blow or force resulting in the
BLOOD PATTERN ANALYSIS

57. II. Directionality of Bloodstain
 It indicates or relates the path droplet followed at the time it impacted the target
BLOOD PATTERN ANALYSIS
Note: The "tail" points to the direction

58. III. Impact Angle Determination
 It is the acute angle formed between the direction of the blood drop and the plane of the
surface it strikes.
BLOOD PATTERN ANALYSIS
Angle

59. III. Impact Angle Determination
BLOOD PATTERN ANALYSIS
Circular
Elliptical

60. III. Impact Angle Determination
BLOOD PATTERN ANALYSIS

61. III. Impact Angle Determination
BLOOD PATTERN ANALYSIS

62. Take 5

63. III. Impact Angle Determination
BLOOD PATTERN ANALYSIS

64. III. Impact Angle Determination
Sin θ = opposite/hypotenuse
i.e. sin θ = width/length of the droplet
e.g. Width = 1.5cm
length = 3.0cm
sin θ= Width (a) 1.5cm / Length (c) 3.0cm
sin θ= 0.5
θ = 30 degrees
BLOOD PATTERN ANALYSIS
How about this?
In a bloodstain measuring 0.04 inches wide
by 4.064 mm long. Determine the angle of
impact.

65.  Breakout room group activity (max 5 in a
group)
 You are required to calculate and determine
the impact angle of blood stains
 You will be provided with the worksheet
 You are given 10 minutes to complete the
worksheet and
 You will present your data to the class
GROUP ACTIVITY

66. IV. Target Surface and Stain Shape
 Bloodstains can occur on a variety of surfaces. The type
of
surface that free falling blood strikes affects the
appearance of the resulting spatter.
 Blood drops on a smooth surface will make a more
uniform
or regular circular shape.
 Blood drops on a rough surface will make an irregular
shaped stain with rough or jagged edges.
BLOOD PATTERN ANALYSIS

67. V. Point/Area of Convergence (POC/AOC)
 The common point, on a 2-dimensional surface (2-D), over which the directionality of
several bloodstains can be retraced.
 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.
BLOOD PATTERN ANALYSIS

68. V. Point/Area of Convergence (POC/AOC)
BLOOD PATTERN ANALYSIS

69. VI. Point/Area of Origin (POO/AOO)
 The point of origin is the location from which the blood that produced the bloodstain
originated.
 It can be constructed graphically by plotting the distance from point of convergence with
their angle of impact on target surface.
 In 3-D.
BLOOD PATTERN ANALYSIS

70. VI. Point/Area of Origin (POO/AOO)
 Can be determined by these methods;
 Stringing method
 Graphic/Graph method
 Trigonometric method (Tangent
formulae)
 Computer programs/ software
BLOOD PATTERN ANALYSIS

71. VI. Point/Area of Origin (POO/AOO)
 Trigonometric method (Tangent formulae)
BLOOD PATTERN ANALYSIS

72. VI. Point/Area of Origin (POO/AOO)
 Trigonometric method (Tangent formulae)
 Tangent of angle of impact= opposite/adjacent
or Z/Y
 Point of origin or Z = Tan of angle of impact x
Y
 Z = Tan of 30 degrees x 15”
 Z = 0.5773 x 15 = 8.66”
BLOOD PATTERN ANALYSIS

73.  Blood is the most common and possibly most important evidence in the solving of certain
types of crimes.
 A drop of blood tells a thousand stories.
 Its COMPLEX!
 Its never simple……creativity is required to solve a crime. We have to look at every
ANGLE!
SUMMARY

74. SUMMARY

75. SUMMARY

76. Check it out! (Blood Spatter)
 https://youtu.be/qFR6JvVJk6o
 https://youtu.be/sCXfosxgyrI
 https://youtu.be/AHH8VItHQvk?si=X
n201zkIPbTLdvTN
 http://www.forensicsciencesimplified
.org/blood/BloodstainPatterns.pdf
REVISION LINKS/RESOURCES

77.  Case #48311
 Discovery of an apparent homicide
 4 deceased, 1-knive, multiple blood stains discovered
 From the crime scene sketch, determine where the incident occurred? Height? Force?
And number of injuries?
REAL-CASE DEPICTION

78. POST-CLASS
In this activity, you will delve into the intricacies of blood analysis at a crime
scene. Your task is to provide an analysis of blood overview of these 3 case
studies. Place your answer in the discussion forum in PB. Do take the time to
discuss other responses too. Thank you!

79. CASE STUDY 1

80. CASE STUDY 2

81. CASE STUDY 3

82. REFERENCES
General information
 Saferstein, Criminalistics: An Introduction to Forensic Science
 Siegel, Forensic Science: The Basics
 Nordby, Forensic Science: An Introduction to Scientific and Investigative Techniques
Presumptive Test
 https://forensicreader.com/presumptive-test-for-blood/
Confirmatory Test
 https://forensicreader.com/confirmatory-test-for-blood/
Blood Pattern Analysis
 https://www.crimescene-forensics.com/Crime_Scene_Forensics/Bloodstains.html

83. CREDITS: This presentation template was created by Slidesgo, and
includes icons by Flaticon, and infographics & images by Freepik
THANKS!
DO YOU HAVE ANY QUESTIONS?
manraj@upm.edu.my
012-5717395
Reminder
Student
evaluation