FORENSIC CHEMISTRY

CHEMISTRY

1. INTRODUCTION

CHEMISTRY DIVISION PHYSICAL SCIENCE

The chemistry division of the PNP Crime
laboratory was established in 1960 under the
name Physical Science Division with the
mission to respond to the need for scientific
examination through the application of
chemical theories and principles in the
examination of physical evidence.
 Functions of Chemistry Division:
     It conducts macro/microphysical, chemical,
instrumentation and comparative examination
of evidence for judicial use and or to aid
investigators in their investigation for the early
solution of the case. It conducts analysis and
comparison of the specimen to determine the
nature, composition, quality or source of the
evidence

     1. Gunpowder determination

a. Paraffin testing
b. Gunpowder residue test
c. Gunpowder range determination
2. Examination of explosives and explosives ingredients & Processing of a bombing
incident
3. Examination of unfair trade competition
4. Conducts research and lectures
5. conduct field laboratory works/crime scene processing
6. Testify in court and give expert opinion were his expertise is required

Functions of Physical Science Division:

1. Hair & Textile fibers

2. Glass & glass fractures
3. Casting & molding of different impression
4. Macro-etching / number restoration
1. Firearms
2. Motor vehicles
5. Soil, mineral & dust analysis
6. Arson or fire debris & accelerants examination
 7. Metals, paints & wood
8. Bullet trajectory
9. Ultraviolet examination
1. Buy bust operation
2. Entrapments operation
10. Conducts lectures & researches
11. Technical opinion in both civil and criminal case

a. Tool marks
b. Shoe/foot marks
c. Tire marks

 Functions of Forensic toxicology:

1. Examination of dangerous drugs & volatile substances

1. Seized drugs
2. Drug testing
3. Processing of clandestine laboratories
2. Alcohol & Blood alcohol determination
3. Examination of poisons
1. Human internal organs
2. Body fluids
3. Food samples
4. Water & gastric contents

Personnel:

1. Forensic chemist
2. Forensic Chemical officer
3. Forensic Analyst

Responsible in applying the natural and applied sciences to the investigation of crimes by
learning to perform laboratory examination of physical evidence submitted in the
laboratory.

2. METHODS USED IN THE EXAMINATION OF PHYSICAL EVIDENCE

METHODS USED IN THE EXAMINATION OF PHYSICAL EVIDENCE

1.Chemicals A substance gives a color a distinct color reaction when brought in contact
with a certain chemical reagents.
 
2.Microscopes
               a. Compound Microscope

          The object to be magnified is placed under lower lens, the lower objective and magnified
image is viewed through upper lens, the eye piece.

                b. Comparison Microscope

          Forensic microscopy requires side by side comparisons of specimen.

It consists of two independent objective lenses joined together by an optical bridge to a common
eyepiece lens.

                  c. Stereoscopic Microscope

          It consists of two monocular microscopes properly spaced and aligned to present a

monocular compound microscope properly spaced and aligned to present a three dimensional
image of a specimen to the viewer, who looks through both eyepiece lenses.

                    d. Polarizing Microscope

          For the study of birefringent materials in which it has a double refraction. The
determination of these refractive index data provides information to identify minerals present in
soil sample or the identity of a man made fiber. 

                     e. Microspectrophotometer

          It is coupled with a light microscope. The examiner studies the specimen under
microscope simultaneously and obtained the visible absorption spectrum or IR spectrum of the
material being observe. 

                      f. Scanning Electron Microscope (SEM)

          It is a soft-ware controlled digital used to produced striking images over a wide range of
magnification (3x300, 000X) on rough or covered surface of minute specimen. It is used for
various forensic examination of trace evidence, such as hairs, fiber, paint particles, drug, metal
and other compound
3.Chromato-graphic technique

                     a. Gas Chromatography (GC)

          It is utilizing for the retention time for the detection of drugs, toxic compounds,
explosives, volatile and semi-volatile compounds.
                      b. Gas Chromatography Mass Spectrometer (GCMS)

          A very powerful tool in the analysis of drugs, toxic substances, explosives, and unknown
compounds. It can be used qualitatively for the identification of a substance present as well as
quantitatively for the determination of the amount of the substance present. This boasts of a
detector capable of identifying virtually unknown substances as it has a built-in library in its
software scrapping the need for standard in most analyses

                      c. High Performance Liquid Chromatography (HPLC)

          An instrument used for qualitative and quantitative determination of the constituents of a

mixture containing volatile and non-volatile compounds by passing through a suitable medium. 

                       d. Enzyme Multiple Immune Assay Technique

          Automated screening analysis of drug of abuse in urine, toxicological serum and body
fluid.
4.Spectroscopic techniques

           a. Ultraviolet

                        Invisible long frequencies of violet light beyond the visible spectrum. 

           b. Infrared

                        Invisible short frequencies of red light in the visible spectrum

           c. Ultraviolet- Visible Spectrophotometer (UV-VS)   

                         Use from qualitative and quantitative analysis of organic and inorganic
compounds containing ultra violet absorbing species and color.     

            e. Fourier-Transforms Infrared Spectrophotometer (FT-IR)

                        It identifies organic substances particularly abused drugs and explosive

ingredients by means of their characteristics functional groups resembling fingerprints of an
individual.
5.Comparative techniques 

1. Identification – The process of determining a substance’s physical or chemical identity.

ex:

1. drug analysis
2. species determination
 3. explosive residue analysis

Comparison - The process of ascertaining whether two or more objects have a common origin.

 A. Individual characteristic:

  It is a property of evidence that can be attributed to a common source with extremely high
degree of certainty.

ex:

    1. matching ridges of finger prints

    2. striation markings of bullet or tool marks

    3. irregular or random wear patterns in tire or foot impressions

    4. comparison of handwriting

B. Class characteristics:

 It is a property of evidence that can be associated with a group and never with a single source

ex:

1. layers of paints
2. blood
3. Hairs & fibers
4. Mass produced products

3. PRACTICE OF FORENSIC CHEMISTRY

PRACTICE OF FORENSIC CHEMISTRY

1.  Collection or reception of Consideration in the collection:

specimen to be examined
a. Sufficiency of samples
      - It is most important that
whenever possible the      Adequate quantities of the specimen should be
chemist/examiner should personally submitted. Even with the best equipment available good
collect all the specimens necessary for results cannot be obtained from insufficient specimens.  
examination.
   

1. Proper collection, handling & b. Standard for comparison

Packing of specimen.
 2. To record the precise position,
make a detailed description, take
notes and make measurements,
sketches and photograph.
3. The chain of custody of specimen
should be maintained.
     Submit a known standard for comparison purposes. If
evidence in question is found in the presence of foreign
substance, a sample of the foreign substances must be
submitted for analysis.
c. Maintenance of individuality
     Each evidence must be collected and preserved as a
separate sample. There must be no mixing or
intermingling of unknown with known samples &
different evidence is separately packed to avoid
contamination

d. Labelling & Sealing

     Mark or label each pieces of evidence for positive

identification.

Information in labeling:

1. Nature & source of sample

2. The date & time of collection
3. Case Number
4. Name of person collecting the sample

2. Actual examination of the specimen  

 a. The first step in the examination of an
article is to scrutinize it carefully and write
down in the laboratory notebook a complete
description of its external appearance
including the manner in which it is secured
and particulars of the sealing. If possible,
photograph the specimen.  The specimen is
opened and any inner wrappings should be
described & detailed description of the
appearances should be taken.
b. The second step in the examination is to
measure or weigh the object.
 A. Qualitative- Identification of a substance
present in a sample
 B. Quantitative-Determining the percent purity of
the sample
1. Physical
     Physical properties describe a substance
without reference to any other substances. It is a
typical property of a particular substance which
can be measured without altering the material
composition through chemical reaction.
   Ex:   a. weight

           b. volume

c. The third step is chemical, microscopically          c. color

or physical testing.
         d. boiling point

         e. melting point

         f. density

         g. refractive index

2. Chemical

      Chemical property describes the behavior of a

substance when it reacts or combines with another
substance.

3. Confirmatory

           Provide absolute identification of the

unknown substances.
3. Report writing of the result of the       1. Heading of the examining laboratory
examination
      2. Report Number
     The result of the examination conducted
will be communicated to the requesting party       3. Case
in the form of a written report which must
include an enumeration of the articles       4. Time & date received
received for examination with detailed
description of the packing, sealing and       5. Requesting party
labeling.
      6. Name of suspect

      7. Name of victim 

      6. Specimen submitted

      8. Purpose of the examination

      9. Findings

     10. Conclusions

      11. Time & date completed

     12. Remarks

     13. Name of Examiner

     14. Name of approving officer

     15. Name of head of office

4.Court duties Expert witness:

     The written report of the                        Qualification:

examiner is usually supplemented
at a later date by oral evidence if                        a.    Education
the case is brought to court or
prosecutors office.
1. The examiner should have a
thorough knowledge of his subject
and of collateral matters relating
to it.
2. All answers to questions and all
opinions should be definite and
free from ambiguity and should be
given in simple language.
1.  Trainings/schooling
2.   Skills/expertise
1. Can state what he perceived and also give his opinion,
deductions or conclusions to his perceptions.
2. Must be skilled in the art, science or trade he is testifying
3. Can testify on things which he has not seen by giving his
opinions, deductions or conclusions on the statements or
facts.
Ordinary witness:

  1. Must have the organ and power to perceive and state

only what his senses perceived
2. May not be skilled on the line he his testifying & cannot
testifying on things or facts he has not perceived
3.  He does not fall in any of the exception provided by law

4. Golden rules in the Practice of Forensic Chemistry

1. Go slowly      Good work cannot be hurried, therefore take all the time necessary to
make the case complete, no matter how urgent it may appear or how
  pressing others maybe for the result; it is generally adjourn a case if the
work cannot be finished in time.
2. Be thorough      Make careful and minute examination of everything and do not be
satisfied with a qualitative analysis if a quantitative examination is
  possible.
3. Take notes      Keep a full, neat and clear record of everything seen and done
4. Consult others      Many cases will lead the expert into paths with which he is not
 familiar and when this happen he should consult those who are most
likely to know.
 
5. Use Imagination      A disciplined imagination which enables inferences and deductions
to make from slender and incomplete premises is often very useful. 
 
6. Avoid complicated      The simplest explanation is usually the right one. The interpretation
theory of results is often the most difficult part of the expe rts work, a precise
knowledge of miscellaneous facts, a sound judgment, good reasoning  
  powers and a disciplined imagination are all essential to success.

Forensic Serology (Body Fluids)

About Body Fluids

1. History of Serology

Many significant advances in the forensic sciences have been made in the field of serology

1800s Early 1900s Mid-1900s

Mid-1800s- Gregor In 1901, a presumptive color
Mendel suggested that change test (Phenolphthalein)
genes control factors was developed by Kastle and
influencing heredity Sheed. This presumptive test
is use tody around the world
In 1916, Leone Lattes of Italy
developed a technique for the ABO
grouping of dried bloodstains. Lattes
were the first to apply ABO blood
grouping reactions to criminal
cases.           

In 1868, Van Deen in In 1901, German scientist
Holland developed a Uhlenhuth developed the
color change test using precipitin test for
an extract from the West distinguishing human and
Indies Shrub quaiacum.- animal blood.
Quaiac test
Likewise, Karl Landsteiner in
Venna disovered the presence
in humans of different blood
groups. This was the origin of
the ABO typing system, a
system that developed into
In 1937, German scientist Walter
Specht developed the chemical luminal
that would “glow” or luminescence in
the dark if sprayed on a bloodstains.
The same presumptive test is widely
used by      crime scene specialist
around the world.
 In 1985, British scientists announce the
development of DNA “  
Fingerprinting”. Alec Jeffreys  and his
colleagues at Leicester University
England were responsible for the
process of isolating and reading these
 most common method of
blood typing
Markers “DNA Fingerprinting”
Researches today uncovers new
approaches and variations to the
original Jeffrefy technique, they use the
term “DNA Profiling” and “DNA
Typing

2. FORENSIC SEROLOGY AND SEROLOGISTS

Forensic Serology Forensic Serologists

-application of immunological and -study blood, semen, saliva and other bodily fluids for use
biochemical methods of analysis to in legal cases.
the identification and characterization
of body fluids in relation to crimes  
and other legal matters
-work on cases involving homicides, rapes, assaults and
 -the use of biological approach to paternity disputes.
law and crime investigation
 
 -Blood analysis is the study of body
fluids; blood serum, saliva, semen -perform laboratory test and interpret body-fluid evidence
and other bodily fluids are all including blood, saliva and semen
included in this area of forensics.
-study crime scene blood patterns, determining directions
  of assault and the distance and angle of a weapon between
victim and suspect.

-determine the time of death based on crime scene blood.

-develop techniques to preserve and ship evidence on

clothing, cigarettes or weapons from the crime scene to
the lab.

-write reports on their findings, and testify on their

analyses in trials as expert witnesses.
3. Definition of terms

1. Antigen A substance, usually protein that stimulates the body to produce antibodies
against it. Substance stimulating the formation of antibodies
2. Antibody A protein that destroys or inactivates a specific antigen. Antibodies are
found in blood serum.
3. Antiserum Blood serum in which there are specific antibodies.
4. Agglutinin An antibody which causes the agglutination of cells containing the
corresponding specific antigen (agglutinogen)
5. Agglutination The clumping together of red blood cells by the action of an antibody.
6. Albumin A major protein constituents of blood serum.
7. Serology The study of antigen – antibody reaction.
8. Fibrinogen A blood protein involved in the phenomenon of blood coagulation.
9. Flourescence The property of producing light when acted upon by radiant light.
10. Precipitin An antibody that reacts with its corresponding antigen to form precipitate.
11. Genetics The branch of biology dealing with heredity and variation in animal and
plants species.
12. Genetic marker A readily recognizable gene which can be used in family and population
studies.
13. Genes A unit of inheritance consisting of a DNA segments located on a
chromosome. It is a basic unit of heredity whi8ch determines the production
of a trait.
14. DNA Deoxyribonucleic acid- the molecules carrying the body’s genetic
information.
15. Chromosomes A rod like structure in the cell nucleus along with the genes located. It is
composed of DNA surrounded by other materials mainly proteins
16. Locus The physical location of a gene on a chromosome.
17. Homogenous The same, composed of similar parts, constant mixture.
18. Homozygous Having two identical allelic genes on two corresponding positions of a pair
of chromosomes
19. Heterozygous Having two different allelic genes on two corresponding positions of a pair
of chromosomes.
20. Genotype The particular combination of genes presents in the cells of an individual
21. Phenotype The physical manifestation of genetic traits such as shape, size and blood
type
22. Dominant gene Gene which is expressed neither they are in the homozygous or
heterozygous
23. Recessive gene Gene which are not expresses except in the homozygous state.
24. Allele Any of several alternative forms of a gene located at the same point on a
particular pair of chromosomes
25. Immune Protected against infection
26. In Vitro Outside the body
27. In vivo Within the body
28. Luminescence The emission of light as a result of external causes other than light. Ex:
ultraviolet radiation
29. Amorph A gene which has no detectable product.
30. Peroxidase An enzyme which catalyses oxidation reactions in which hydrogen peroxide
is an electron acceptor
31. Enzyme A protein substance produced by living cells capable of speeding up
chemical transformation or reaction, such as oxidation.
32. Saliva - Watery fluid secreted by submaxillary, sublingual, parotid and buccal
glands   the mouth. Containing proteins, inorganic substances and enzyme.
33. Salivary The variety of starch hydrolyzing enzyme present in the saliva.
amylase
34. Titer The effective strength of concentration of antibody in an antiserum.
(antiserum)
35. Specificity The property of an antibody that enables it to combine with one but no other
antigen

4. Types of Bodily Fluid

Body fluids are broken down into two categories: excreted and secreted.

Excreted:          Secreted:
Sweat, Breast Milk, Cerumen (Earwax), Faeces Cowper's Fluid (Pre-ejaculatory fluid),
(included because faeces are often covered in a mucus Blood or Plasma, Semen, Saliva,
membrane to enable travelling through the bowel), Female Ejaculate, Serum, or Urine.
Chyme (found in the stomach), Bile, Vomit, Aqueous
Humour (a watery substance that covers the eye), Sebum
(Skin Oil)

5. Types of physiological Evidence

1.Blood

            -is the most common physical evidence found at the scene of a crime.

             -It can be deposited in the form of trace amounts, drops, smears or small pools.
2. Semen

            -are the next most common type of physiological fluid encountered in the investigation
of crime, usually in connection with sexual assault and rape cases.

            -When wet, semen is grayish-white in color and bears a Clorox like odor.
3. ***l secretions

           -are a complex mixture of cells and secretions.

           -screening tests based on microscopy have been proposed.

           -***l epithelial cells are large, and many contain glycogen which can be demonstrated by
staining with iodine in the form of a solution or exposing to iodine vapor. 

          -the presence of markers that are also used to type semen, specifically ABH and PGM1.
          -It is not possible to distinguish grouping results by physiologic origin with an acceptable
degree of reliability. 

          -This includes situations where the woman is a non-secretor and the man is a secretor

***l Smears

          -When the swab is fully inserted, rotate the end through 2-3 revolutions, which will allow
the cotton tip to pick up an adequate load of cells.

          -The swab can then be gently withdrawn.

          -Prepare the smear immediately after withdrawal of the swab by rolling (not sliding or
rubbing) the cotton tip along the length of a glass microscope slide.

         -Generally, two parallel tracks can be rolled on a single slide.

         -Diff-Quik is a very convenient stain for use with ***l smears.

         -It consists of two solutions: an eosinophilic (red) solution and a basophilic (blue) stain.

        -To stain the smear, dip the slide in and out of the red stain 5 to 10 times, the in and out of
the blue stain 5 to 10 times.

         -Presence of  ***l epithelial cells.

         -leukocytes, erythrocytes and bacteria are usually evident

         -small numbers of other contaminating cells and microorganisms are sometimes observed.

         -Parabasal cells are the smallest epithelial cells seen on a typical ***l smear.;are round or
nearly round and have a high nuclear to cytoplasmic ratio.

          -cells are typical intermediates except for the one cell in the middle panel (arrow), which
might be classified as a superficial cell (small, dark nucleus).

Note that the intermediate cells vary in size and that some have rounded outlines (small
intermediates), while others have a polygonal shape (large intermediates
4.Saliva

          -fluid that moistens the mouth.

          -secreted from three sets of glands – the sublingual, submandibular, and parotid. 

          -saliva from the parotid glands contains amylases, which aid in the digestion of
carbohydrates

          -Screening is based on detection of high levels of amylase in the sample.

          -not a confirmatory test as amylase is found in other body fluids.

          -contains ABH substances, especially in secretors. 

          - Serous secretion is a more liquid opalescent fluid composed of water and proteins, such
as the digestive enzyme amylase

         -rich source of DNA and buccal swabs for  DNA typing.

         -As evidence, samples are collected from suspects and victims

         -evidence in sexual offenses where oral contact is alleged, bite marks, or on cigarette butts
discarded at a scene, cups, glasses, toothpicks, and other items placed in the mouth.

          -samples (spit or buccal swabs)  for determination of secretor status. 

          -Stains can be typed using absorption-elution or absorption-inhibition.

5.Urine

          -contains a large amount of urea, a chemical byproduct of normal metabolic processes in
the body.

          -Identification of high levels of urea can therefore serve as a screening test for urine in
fluids or stains. 

          -The presence of creatinine is also used for screening purposes

          -Creatinine forms a red compound with picric acid (known as the Jaffe Test).

          - Urine also has a characteristic odor, which can help in locating its presence.

          -Gentle heating of urine-stained materials gives rise to a distinctive odor.

          -Urine from secretors will contain ABH substances.

6.Feces

          -food residues passed from digestive system. 

          -characteristic odor mainly due to skatole.

          -Laboratories may be requested to test stains or other samples for the presence of feces.
          -Investigation of anal intercourse or where perpetrators have fouled a crime scene.

         -Screening of samples depends on the detection of urobilinogen, a bile pigment excreted in
feces.

         -detected using its fluorescent reaction to Edelman’s reagent.

7.Vomitus

          -is not frequently encountered at the crime scene.

          -it can provide some limited information for the investigator and crime scene specialist if
present.

          -Aside from any laboratory testing, it may reveal some clues regarding the time and
content of the contributor’s last meal.

          -It may provide clues about the person’s medical condition

BLOOD AND BLOOD STAINS

BLOOD AND BLOOD STAINS

1. BLOOD

- It refers to a highly complex mixture of cells, enzymes, proteins and inorganic substance.

- Red fluid circulating throughout the body via the heart and lungs to transport oxygen,
nutrients and wastes.

- The red liquid that is circulated by the heart and flows in the veins, arteries and   
capillaries.

2. FUNCTIONS OF BLOOD

1. Nutritional It supplies tissues throughout the body with food materials and substances
absorbed from the gastro intestinal tract.
2. Respiratory It carries oxygen from lungs to tissues and carbon dioxide from body tissues to
lungs.
3. It helps protects the body against infection by the phagocytic activity of certain
Immunologic- white blood cells and by the production of proteolytic enzymes and antibodies
Body defense in the blood stream.
mechanism
4. Maintenance It assist in the preservation of an almost neutral reaction in the tissues by its
of selective excretion of soluble substances and its buffering power. The
homeostasis maintenance of a normal water balance and fluid distribution throughout the
/buffering body depends on the mobility of the water contained in the blood.
action
5. Excretory It carries waste products of catabolism of the tissues to the main excretory
organs, the lungs and kidneys for elimination
6. Blood coagulation/blood clotting
7. Regulation of - It maintains organs of the body within closely restricted limits of temperature.
temperature The metabolic processes which occur during cell activity produce heat and
blood tends to minimize even minor variations in local temperature as it passes
through capillaries of the different body organs. The circulation of blood in
vessels in the skin and lungs also enables heat to be lost from the body by
radiation and evaporation.
8. Transportation of hormones and other endocrine secretions that regulate cell functions

3. PHYSICAL CHARACTERISTIC OF BLOOD

1. In vivo, it is in fluid form because of naturally occurring anticoagulant but in vitro, it

coagulates within 5-10 minutes.

2. It is red in color due to hemoglobin

3. It is slightly alkaline with an average ph of 7.4

4. It has an average specific gravity of 1.055

5. It is thick and viscous; 3.5-4.5 times thicker than water

6. It makes up 7-8% of the total body component or 75-85 ml blood per kilogram body
weight.

7. There are approximately 20 gm solid per 100 bloods.

4. ANTICOAGULANTS

It is a chemical preparations added to blood to prevent clotting.

1. In vivo, it is in fluid form because of naturally occurring anticoagulant but in vitro, it

coagulates within 5-10 minutes.

2. It is red in color due to hemoglobin

3. It is slightly alkaline with an average ph of 7.4

4. It has an average specific gravity of 1.055

5. It is thick and viscous; 3.5-4.5 times thicker than water

6. It makes up 7-8% of the total body component or 75-85 ml blood per kilogram body
weight.

7. There are approximately 20 gm solid per 100 bloods

5. METHOD OF BLOOD COLLECTION

1. SKIN PUNCTURE 2. VENIPUCTURE

It is used when small quantities of blood are
required
It is a collection of blood from puncture made
on skin
It is the easiest and most convenient method of
obtaining enough volume of venous blood
suitable for a variety of tests:
Factors involved in venipuncture:

blood obtained is known as: 1. Venipuncturist

A. Capillary blood                                                - Phlebotomist motto: the first thing is not to

inflict damage
B. Peripheral blood
2. Patients and his veins
C. Arteriolar blood
in adults;                
 
       1. Wrist veins

       2. Veins dorsal portion of the hand

       3. Veins on antecubital fossa

3. Equipment
6. COMPOSITION OF BLOOD

Formed elements( 45%) Liquid Gaseous

portion( 55%)
1. RBC- erythrocyte, normocyte, erythroplastids Serum 1. Oxygen
2. WBC- leukocytes, leukoplastids Plasma 2. Carbon dioxide
3. Platelets-(thrombocytes)    
7. Three Cellular Components of Blood

1. Red blood cells (RBCs) or 2. White blood cells (WBCs) 3. Platelets or thrombocytes
erythrocytes or leukocytes
-known as erythrocyte, 1. Granulated WBC cells
normocyte and erythroplastids
a. Neutrophils
-predominant formed
elements b. Eosinophils

- Composed of  hemoglobin, c. Basophils

the oxygen transport material
2. Agranulated WBC cells

a. Monocytes

b. Lymphocytes
Serum Plasma
It is the liquid portion of clotted blood without It is the liquid portion of unclotted
fibrinogen since clotting is due to the polymerization of blood with the protein fibrinogen. It is
the plasma proteinfibrinogen into fibrin. When whole obtained when fresh blood is mixed
blood coagulates , the cellular elements are trapped in with an anticoagulants and then
the fibrin mesh. Upon standing, the clotted blood centrifuged or allowed to stand until
undergo retraction separating from the wall of the the cells have settled.
container and shrinking in volume, threby squeezing out
a straw colored fluid known as serum.
HEMOGLOBIN
HEMOGLOBIN

1. HEMOGLOBIN

- A red blood cell protein responsible for transporting oxygen in the blood stream and the
red coloring of the blood.

2. Types of Hemoglobin

Normal or functional Abnormal or non functional

1. Oxyhemoglobin (Hbo2)- Arterial blood-
bright red in color
2. Deoxyhemoglobin or reduced
hemoglobin (HbCo2)- Venous blood- dark
red in color
3. Carbon monoxide poisoning
1. Carboxyhemoglobin ( HbCO)-cherry red in
color
2. Sulfhemoglobin (Shb)- lavender in color
3. Methemoglobin (Hi)- Hb
    Other terms: Ferrihemoglobin,Oxidized
hemoglobin,  Hemiglobin-  chocolate red in color
-occurs when gas is inhaled

-toxic gas, which is colorless, odorless, tasteless, and initially non-irritating, it is very difficult
for people to detect.

- product of incomplete combustion of organic matter due to insufficient oxygen supply to

enable complete oxidation to carbon dioxide (CO2).

- Result is headache, nausea , convulsions, and finally death by asphyxiation

- In pathological (autopsy) examination:

-unembalmed dead persons are normally bluish and pale.

-dead carbon-monoxide poisoned persons may simply appear unusually lifelike in

coloration-looking pink-cheeked and healthy.

-Breath CO monitor displaying carbon monoxide concentration of an exhaled breath sample

(in ppm) with its corresponding percent concentration of carboxyhemoglobin.

4. Methemoglobin

-is a form of the oxygen-carrying metalloprotein

hemoglobin, in the iron  heme group ( Fe3+ (ferric) state.)

-cannot bind oxygen.

-bluish chocolate-brown in color.

In human blood a trace amount of methemoglobin is

normally produced spontaneously.

-But when it is present in excess the blood becomes

abnormally dark bluish brown.

-The NADH-dependent enzyme methemoglobin reductase

(diaphorase I) is responsible for converting methemoglobin
back to hemoglobin.

- The most famous "blue man" -- known as "Papa Smurf"

-- has died.

-Paul Karason was born a fair-skinned, freckled boy with

reddish blond hair.

-But later, he developed skin with a bluish tinge against his

shock of white hair,

-the result of a rare medical syndrome known as argyria or

silver poisoning from dietary supplements.
COLLECTION, PRESERVATION AND TRANSMITTAL OF
EVIDENCE
COLLECTION, PRESERVATION AND TRANSMITTAL OF EVIDENCE

1. COLLECTION, PRESERVATION AND TRANSMITTAL OF EVIDENCE

Blood and other body fluids can be examined for species, race, sex, type, DNA, and other
characteristics. Comparison of questioned to known is also possible. The following
guidelines for the collection and preservation of questioned blood, body fluids and tissues
are included here as a general reference; the investigator should get precise instructions on
handling serological evidence from a certified crime scene technician or from the serology
section of their jurisdiction's forensic laboratory.

1.Document any body fluid patterns, spatters, stains, pools, drops, and the like with close-up
scaled photographs and medium distance context-establishing shots.
2. Any item or material bearing suspected blood, semen, saliva, other body fluid stains or tissues
must be allowed to air-dry at room temperature prior to packaging; exposure to direct sunlight
and/or heat should be avoided. Failure to ensure complete drying of such samples may result in
their putrefaction and loss.
3. Use cleaned tweezers or other tool to remove the body fluid-stained item. Package each item
individually in an air-permeable but otherwise securely closed container such as a paper bag.
Fragile substrates such as glass should be carefully wrapped in paper and securely packages to
avoid (further) breakage.
4. Label and seal the container properly, including your name, date, description, and exhibit
number. Consult your jurisdiction's forensic laboratory or a certified national laboratory for
instruction on refrigeration of samples.
5. If the stain is on a substrate which is too large to submit intact, either cut away the section of
the surface bearing the stain, or dismantle the object to recover and submit the stained portion.
Collect according to steps 2 through 4.

Sufficiently copious bloodstains on an immovable substrate may be scraped off with a clean
knife or razor blade and collected into glassine or clean white paper, folded pharmacy-style to
prevent leakage, and placed into a paper envelope. Do not collect scrapings directly into
envelopes. If you are collecting multiple samples, each must be collected with a clean tool.
Label the container(s) according to step 4. Retain and package the tools used to collect the
scrapings.
6. If the stain is encrusted on the surface of soil or sand, remove the crusts and place into
separate paper pillboxes; then collect the bulk stained matrix in paper ice cream-type containers.
If moist blood is available for collection, such as from a pool on a tile floor, use a clean dropper
to collect as much as possible (up to 10 cc) into a glass vial. Add an equal volume of isotonic
saline solution (0.9% sodium chloride) to the vial. Seal and label the vial as described above. Or,
you may soak up the moist blood with a new, sterile gauze pad, air dry the gauze pad, then
collect the gauze pad as described above in steps 2-4. Biohazardous material should be marked
as such before it is sent to the lab
PHASES OF LABORATORY EXAMINATION 1
During the examination of evidence for the presence of blood it is often convenient if not
necessary to employ a screening test. Such test would be followed with a confirmatory test
before a conclusive identification were made. The following procedure details several of the
more common screening and confirmatory tests used. In exercising these procedures it
cannot overemphasized that while a negative screening tests calls for no further
examination, while a positive test virtually requires a confirmatory test before a stain may be
identified as blood. Many of the techniques and procedures used by the forensic serologist
involve complex biochemical reactions. A number of basic testing procedures utilize
chemical principles in the determination of blood.

1. PRELIMINARY TEST FOR BLOOD

A. BENZIDINE TEST – (Alder reaction)

B. PHENOLPTHALEIN TEST- Kastle Meyer test

C. ORTHO-TOLIDINE TEST (Guaiacum test, Van deen dya’s or Shombein test)

D. LEUCOMALACHITE GREEN TEST

Screening test for Bloodstains

. A. BENZIDINE TEST – (Alder reaction)

        The oxidation of benzidine provides a highly sensitive and convenient presumptive test
for the presence of blood. The test is quite sensitive (1 part in 300,000 to 500,000) but must
always be considered a screening test for blood. A negative test is conclusive evidence of
the absence of blood in quantities sufficient for further examination. Insolubility in a
bloodstains can be a limitation with some evidence, and in this case a single thread may be
removed and the test solutions applied directly to the thread. Another limitation may be
occurrence of a false positive reaction due to substances other than heme which possess a
similar peroxidase- like activity or to materials containing peroxidase itself.

ADVANTAGES:

     The test never fails to detect blood even when very old and decomposed stain.

Chemical Reagents: Procedure Result

a. Benzidine solution (small
amount of benzidine
dissolved in    glacial acetic
acid)
 
a. Place a small fragment or portion
of the stained material on a filter
paper.
b. Add a drop of benzidine solution
on a stained material
An intense blue color
radiating out from the
source of the stain will
produce immediately if
blood is present

b.3% Hydrogen peroxide c. Add a drop of  3% hydrogen

solution peroxide solution
3. B. PHENOLPTHALEIN TEST- Kastle Meyer test

         Like the Benzedrine test, this test is a presumptive test for the presence of blood and
requires a confirmatory test for a conclusive identification. It is considered more sensitive
than the Benzedrine with literatures values from 1:1000, 000 to 1:10,000,000. This sensitivity
is great enough to cause some authors to advocate boiling 1-2ml of a saline suspension of
the suspected blood for a half a minute to destroy any oxidases that might be present. The
reagents are added directly to the suspension to perform the test. This treatment, however,
can only be expected to destroy plants peroxidases, as they are rapidly inactivated at 100oC
while animal peroxidases are not. Boiling, then, is only limited value. It can be shown to give
a false positive reaction with number of oxidizing agents, plants and animals substances as
those under the “Benzidine Test solution”.

DISADVANTAGES:

            The disadvantages of the phenopthalein test is the viability of the prepared reagent
which must be kept in a dark bottle and include the procedures in the preparation of the
reagents.

Sources of a False Positive Reaction (Benzidine &Phenolphalein Test)

Apple Bone marrow Rust
Apricots Leukocytes Formalin
Asparagus Brain Tissues Soil
Beans Spinal fluid Copper Sulfate
Beets Intestine Potassium Permanganate
Black berries Liver Potassium Dichromate
Jerusalem antichoke Lungs Bleaches
Horse radish Saliva/Sputum Clay
  Mucus/pus/nasal secretions  
 

Chemical Reagents Procedure Result

a. Phenolphthalein solution ( 1 to 2 a. Place a small fragment of the A pink color
grams of  phenolphthalein to 100 ml of stained material on a filter paper will be produced
25% potassium hydroxide in water if blood is
added with one gram of zinc powder b. Add a drop of phenolphthalein present
heated until colorless) solution to the stain

b.3% Hydrogen peroxide solution c. Add a drop of  3% hydrogen

peroxide solution
4. C. ORTHO-TOLIDINE TEST (Guaiacum test, Van deen dya’s or Shombein test)

 This is not widely used procedures as a screening test, the O-tolidine test is

gaining popularity probably due to its great chemical similarity to Benzidine test and the
increasing in availability of benzidine. The chemistry of the test is essentially the same as
that of the benzidine test.

Chemical Reagents Procedure Result

a. Fresh tincture of guaiac resin
(few lumps of guaiac resin in
95% ethanol, then filter)
b.3% Hydrogen peroxide
solution
5. D. LEUCOMALACHITE GREEN TEST
a. Place a small piece of stained
fabric on a porcelain dish
b .Soak with fresh tincture of
guaiac.
c. Add a few drops of 3%
hydrogen peroxide solution
An intense blue color
radiating out from the
source of the stain will
produce immediately if
blood is present

     LMG, the reduced form of the dye, provides another screening test for the presence of
blood. Like the other screening test discussed, it is an oxidation-reduction system and can
only be considered as an indicator for further confirmatory testing.

Chemical Reagents Procedure Result

a. Leucomalachite Green solution a. Place a small amount of the deep green or malachite
( 1 gram leucomalachite green stained fabric on a fabric paper. green color/pea** green
dissolved in 48 ml Glacial acetic color
acid and diluted to 250 ml with b. Add a drop of leucomalachite
green solution on the stained
water.) fabric

b.3% Hydrogen peroxide c. Set aside for a few seconds and

solution then drop 3% hydrogen peroxide
solution
6. Screening test for Bloodstains

1. Luminol   

-In 1928 Albrecht reported on the chemiluminescent properties of the compound 3-

aminophthalhydrazide.

 - In 1935 Hundress, Stanley, and Parker published a method for the [reparation of the
compound and named it luminol.

 -In 1937 Specht published his findings on the application of luminol to the forensic field

 -It was through his studies that the use of luminol as a chemiluminescent test for the presence of
blood was advocated.

 -it is generally recognized that two tautomeric forms of the oxidized structure create the
chemiluminescent qualities

-Once sprayed on a surface, a bluish light emission may be detected at concentrations as low as
0.1 PPM.

- Luminol is employed when no visible blood is detected or other less sensitive presumptive
tests have failed.

-It is also primarily used for large items such as cars and houses.

 -false positives will occur on vegetable peroxidases, some metals and chemicals.

-Luminol reactions must be photographed in total darkness so that the only light to strike the
film comes from the luminol reaction.
 

2. Fluorescein test

 -the chemical mixture of fluorescein causes a catalytic reaction to occur between

the hemoglobin in blood and oxygen.

-produces a luminescent stain, which will luminesce in the dark when excited by Alternate Light
Sources (ALS).
-major advantage is that it will continue to luminesce for hours under UV or ALS after the initial
application, and without additional applications of the reagent
 

3.Leucocrystal Violet (LCV)

-is a relatively new positive blood identifier that turns permanently violet when in contact with
blood.

- Leucocrystal violet can be used in testimony in court as a blood identifier at the scene without
the need for further testing to identify the stain as blood
           

4.Tetra-Methyl Benzidine (TMB)

-presumptive tests that is specific for blood.

-is an enhancement reagent.

-reacts with the HEME in the blood.

-spray the surface lightly two to three times.

-bloody imprint pattern should turn a greenish-blue.

-overspray may give a very dark blue pattern and mask ridge patterns.
PHASES OF LABORATORY EXAMINATION 2
PHASES OF LABORATORY EXAMINATION

1. 2. CONFIRMATORY TEST

Microscopic Micro-crystalline Spectroscopic Biological

1. Wet method 1. Teichman- hemin 1. Recent stains- a Precipitin test-Hecktoen
test- large dark recent blood stains test
a. RBC rhombic crystals gives a solution of
oxyhemoglobin
b. WBC
2. Stained preparation 2. Takayama- 2.Old stains-  Result:
haemochromogen test- oxyhemoglobin is 
a.RBC dark salmon pink color converted to a. Precipitation- ring
methemoglobin due to formation or cloudy
b.WBC-neutrophil, exposure to light  and appearance
eosinophil, air.
lymphocyte, b. No Precipitation- no
monocytes, basophils ring formation/cloudy
- examinations of 3. Acetone-wagenhaar -most delicate and - The simplest and
avian, piscine, test- small dark reliable test for probably most common
menstrual, nasal, rhombic crystals determining the technique of origin
lochial blood, presence of both old determination available to
parasites and blood and recent stains. the forensic serologist
abnormalities
 
2. 1. TEICHMAN OR HAEMIN TEST

-Hemoglobin is readily separated into its protein and prosthetic group components by
treatment with acid. The oxidation of the iron of the heme group takes rapidly in an acid
solution.

LIMITATIONS:

1. The test given by indigo-dyed fabrics not stained with blood so that in case of doubt, a
drop of hydrogen peroxide is added to the crystals and if haematin is present, bubbles of
gas will be given off.

2.  If the stain is old, washed or is changed by chemical agents, the crystals are not formed.

3.  The addition of too much salts, or presence of moisture in the acid or over heating of the
slide may result in failure.

Chemical Reagents Procedure Result

Potassium chloride a. Extracted blood sample is placed on a glass Presence of dark
slide rhombic crystals in
Potassium bromine singles or clusters
b. Teichman reagent is added
Potassium iodide
c. Heat the sample gradually. Allow to cool
Glacial acetic acid
d. Examine under the microscope
 

Teichman test

-confirmatory test for blood based on the formation of distinctive hematin crystals that are
viewed under a microscope.
-developed in 1853 by Ludwig Teichmann and is identified with both the Teichman and
Teichmann spellings.

-referred to as crystal, micro-crystal, or microcrystalline tests.

-Crystal tests require a larger sample than do the presumptive tests for blood and are susceptible
to interferences.

-for very small stains, the crystal test is not used since it will consume material most likely
needed for DNA typing.

-the hemoglobin of blood reacts with acetic acid and sodium chloride to form linear and
rectangular crystals.

-confirms whether a sample is blood, based on the appearance of Teichmann Crystals

-crystals are formed by the reaction of the hemoglobin in blood with the glacial acetic acid and
sodium chloride.

-hemoglobin appears to gain chloride ions and thus transform into a crystal.

-crystals were dark red and orange as well as long and rhombic shaped.

-Some undissolved salt crystals can be viewed under the microscope

3. II. HEMOCHROMOGEN (TAKAYAMA TEST

    This is a delicate test for the presence of haemoglobin. It has long been a method of
choice of confirming the identity of a bloodstain, numerous workers have tried various
modification of the reagent. The test is more sensitive and simpler to perform than the
hemin test and false positives are no problems.

RESULT:

    Large rhombic crystals of salmon pink color which is arranged in clusters, sheaves and

other forms.

Chemical Reagents Procedure Result

Saturated aqueous glucose 1.Extracted blood sample is placed on a Presence of dark
solutions glass slide rhombic crystals in
singles or clusters
10% aqueous sodium
hydroxide 2.Teichman reagent is added

10% pyridine 3. Heat the sample gradually. Allow to

cool
Distilled water
4.Examine under the microscope
 

Takayama test (Confirmatory Testing)

-confirm the presence of blood and is designed to be used in conjunction with presumptive
testing for blood.

-Hemoglobin is composed of a heme prostetic group and globin.

When a blood stain dries, the iron in the heme group is in the ferric state (FE+3) due to the
formation of methemoglobin.

Procedure:

 the dried stain must be hydrolyzed to metheme and globin via alkaline hydrolysis by sodium
hydroxide

-the iron in the ferric (Fe +3) state must be reduced to the ferrous state (Fe +2) via the use of
glucose, a reducing sugar.

-in the ferrous state, the iron will combine with pyridine to form pyridineferroprotoporphyrin,
which is an insoluable crystalline product.

-based on the formation of hemochromogen by heating a dried stain in the presence of pyridine
and glucose under alkaline conditions.

-Result:

A positive result is visualized microscopically by the formation of salmon colored rhomboidal

or stellate crystals

False Positive reaction

only materials that will give a positive reaction other than blood are commercially produced
preparations of catalase and peroxidase, items not occurring in nature.
4. III. ACETONE-HAEMIN OR WAGENHAAR TEST

-       Oxidation of hemoglobin

Chemical Procedure Result
Reagents
1.Acetone A presumptive test for blood in which a small amount of Small dark,
acetone (propenal) is added to the bloodstain, followed by a dichroic, acicular
2. Oxalic acid drop of hydrochloric acid. crystals of
acetone-haemin
 Haemoglobin produces derivatives such as haematin and
haemin, forming small characteristic crystals that can be
identified under a microscope.
 

Spectroscopic examination Another spectroscopic test

-spectroscopic methods, such as ultraviolet- -based on the fluorescence of hematoporphyrin
visible (UV-VIS) absorption, are considered from excitation with ultraviolet light.
highly reliable in confirming the presence of
blood in a stain.  

-many different derivatives of hemoglobin have -successful with bloodstains on oxidized metals,

a characteristic strong absorbance band around bloodstains up to ten years old, and bloodstains
400 nm that is called the Soret Band. exposed to conditions not suitable for UV-VIS
absorption such as heat, sunlight, and humidity.
-can distinguish between
different hemoglobin derivatives and also show  
a positive result for older stains that have a
negative result with other presumptive tests and -Best results can be obtained by analyzing a
crystal tests. saline extract of the bloodstain, and it is
important to keep in mind that there are
-conditions which can interfere with the possible false positives since porphyrin
spectral results such as water submersion, compounds occur frequently in biological
sunlight exposure, heating, and rust. systems.
 

Human (or Species) Origin test - via Ring Precipitin, Ouchterlony, or Anti-
human Hemoglobin Test Methods

1.Precipitin test

-is one method of distinguishing between human and animal blood.

-utilizes the biological properties of antibody-antigen complex formation to allow a visual

representation of a reaction.

-Antibodies are very large molecules and are represented by five classes of immunoglubulins,
IgG, IgA, IgM, IgD, and IgE.
-An antigen is a substance which has the ability to produce an immunological response when
introduced into a foreign animal

-antibodies are produced by a host animal when the animal is injected with a foreign protein
containing antigens and the host becomes sensitized.

-immune system of the host recognizes the foreign antigen and produces antibodies to react with
it in a very specific manner.

-In common forensic testing, the antibodies to human antigens are raised in rabbits which results
in rabbit anti-human antiserum.

-Dr. Uhlenhuth in 1901, presented evidence of the specificity for human antigens to only
agglutinate with complementary antibodies.

-if the antibodies in the anti-human antiserum comes in contact with human antigens, the
specificity of the reaction allows for the formation of the human

-recover these antibodies by bleeding the animal and isolating the blood serum

-serum will contain antibodies that specifically react with human antigen   (human antiserum).

-drop of this anti-human serum is added to suspect blood.

-it will precipitate its protein if it is of human origin.

the precipitin test is sensitive, and will work on small traces of blood

-Precipitin is now a generic name for the resulting agglutinated complex formed when
antibodies present in the serum of a species agglutinate the proteins in the blood of a different
species.

-The forensic test consists of collecting the blood sample in a test tube containing serum from a
rabbit containing antibodies against human blood, known as anti-human antibodies.

‘-If an insoluble complex of precipitin (clumping) occurs, the test is positive for human blood

Species Origin

Human antiserum will only attach to human blood sample

Rabbit antiserum will only attach to rabbit blood sample

Dog antiserum will only attach to dog blood sample

 
2.Ouchterlony Double Diffusion

-most species identification uses radial diffusion of antigen and antibody through agar gel

-Stain and controls samples are loaded in the outer wells and a drop of anti-human antiserum is
loaded into the center well.

-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).

-The plates are left at 4°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 or other general protein stain, to enhance sensitivity and clarity

 
 

3.Crossed-Over Electrophoresis

-test can also be conducted using gel-electrophoresis, when a blood sample is put on a glass
slide and covered by a layer of agar gel.

-uses an electric field rather than diffusion to move the extract and antibody through the gel.

 -slide is positioned side by side with another containing the rabbit anti-human serum, inside a
box filled with a solution that conducts electric current.

-As the current passes through, protein molecules are filtered into the gel and toward each glass
slide.

-If precipitin is formed, the test is positive, and the blood sample is identified as human blood.