Identification from trace substances

By dr. Naveen

• Every criminal carries some elements with him from the scene of crime by which he can be linked with the crime. Edmond Locard (1877-1966) • Every criminal leaves some elements from him to the scene of crime by which he can be linked with the crime. Zhu Yunliang

• • • • • • semen stains hair saliva stains Bone Tissue blood stains


Importances of Examination of blood
• 1. identification of the victim or offender of a crime like homicide, sex offences or where death occurs due to rash or negligent acts on the part of persons who are expected to act with responsibility. Apart from identification of individuals, examination of blood may settle other issues in criminal cases like, to know the the cause of death; • 2. The cause of death (e.g. detection of poison, or some other pathology, responsible for the death) ; •

Importances of Examination of blood
• 3. time of death (different chemical and biochemical tests of blood may be helpful) ; • 4. criminal abortion cases; • 5. investigation of sex offence cases; • 6. to establish relationship between the offence, offender, the offended (victim of the offence) and the offending agent; and, lastly, 7. The malingering cases.

To answers the question of:
• 1 Whether the stain is due to blood or some other material ? • 2 If it is due to blood, then whether it is of human origin or it belongs to some other animal? • 3. What is the source of the blooding: • a) Is it from arterial or venous source ? • b) Does it belong to the victim or the accused ? • c) Is it from an injury, or due to haemoptysis, • haematemesis, menstruation or miscarriage ?

4. Who left the blood?(personal identification) 5. In connection with the cause of death, it should be seen whether the blood contains any poison in lethal concentration or there is any other abnormality which can be considered as the cause of death, as in case of mismatched blood transfusion or heamopoetic disorder. 6. In case of death, time passed after death. 7. Time passed after bleeding, i.e., time of assault or injury (known from the age of the stain, whether recent or old) 8. Whether the bleeding was antemortem or postmortem.

Blood Stains
• Blood that is in liquid pools should be picked up on a gauze pad or other clean sterile cotton cloth , air dry thoroughly at room temperature. It should be refrigerated or frozen as soon as possible and brought to the Laboratory as quickly as possible. Delays beyond 48 hours may make the samples useless in serological analysis.

∀ If close to the Laboratory, deliver the samples immediately. ∀ If unable to deliver to the Laboratory, or if the object must be mailed, allow the stain to air dry completely before packaging.

Do not heat the samples or place it in bright sunlight to dry. Hang clothing and similar articles in a room where there is adequate ventilation.

• Label all samples bits and package each samples separately. If damp, allow fabric to dry completely before packaging. • Handle fabrics as little as possible.

Dried Blood Stains
∀ On clothing, if possible, wrap the item in clean paper, place the article in a brown paper bag or box and seal and label container. Do not attempt to remove stains from the cloth. ∀ On small solid objects, send the whole stained object to the Laboratory, after labeling and packaging.

∀ On large solid objects, if impractical to deliver the whole object to the Laboratory, scrape the stain onto a clean piece of paper, which can be folded and placed in an envelope. Do not scrape directly into evidence envelope. Scrape blood from objects using a freshly washed and dried knife or similar tool. Wash and dry the tool before each stain is scraped off. Seal and mark the

1. Naked eye examination
In naked eye examination, non-coagulated blood is reasonably thick, viscid, and may be bright red or slightly dark red in color depending on whether it is from arterial source or from venous source, respectively. A blood stain is reddish and soft in case of recent bleeding. It is dark red or even blackish, if old. Bleeding from arteries has a sprouting effect (jet like ejection ) and is bright red when fresh. Bleeding from a vein occurs passively, in drops. The drops may have stellate appearance. Even when fresh, it is rather dark.

Jet like ejection ( bleeding from arteries)

2 Chemical tests
These r Exclusionary tests 1. Benzidine Test sensitive , one part from three million.Benzidine is known as carcinogenic 2. Phenolphthalein Test (Kastle-meyer test) 3. Ortho-tolidine test

Benzidine test
cut out a small piece of stained material or tease out fibres from the stained fabric and place it on a porcelain tile.Add a drop of saturated solution of benzidine in a glacial acetic acid then a drop of 10 volumes hydrogen peroxide.If the blood is present, drak blue color is produced immediately. a positive reaction is given by blood of almost any age, blood that has been exposed to heat or cold, blood stains treated with cleaning agents.

The test is very sensitive(positive with 1 in 1,000,000 dilution) but not specific. any organic substance that liberate nascent oxygen from the hydrogen peroxide can produce false positive results. when a negative result is got, then there is no necessity to go for any other test, for example, confirmatory test.

Phenolphthalein test
• Also called Kastle-Mmeyer test to a solution extracted from the stain with distilled water, add 10 to 20 drops of phenolphthalein reagent[ phenolphthalein 2 gm , sodium hydroxide 20 gm, distilled water 100 ml] and then a drop or 2 drops of 10 volumes hydrogen peroxide.If blood is present a pink or purple color develops immediately.

Ortho-tolidine test
• To a solution extracted from the stain add 2 to 3 ml of 4 percent ortho-tolidine followed by a few drops of hydrogen peroxide.Blue color appears if blood is present.

3. Microscopical and Microchemical tests
• These are conformatory test
1. Takayama Crystal Test 2. Teichmann Crystal Test 3. Spectroscopic Test

Takayama crystal test
• Place a small piece of suspected material on a glass slide add 2 to 3 drops of takayama reagent[ sod.hydroxide, pyridine,glucose] and cover with a coverslip.Pink, feathery crystals of haemochromogen or reduced alkaline haematin arranged in clusters, sheaves, etc. appear in one to six minutes.Slight warming of the slide hastens the reaction.The result is negative if crystals are not formed in half an hour. The test give good result even with old blood.

Teichmann’s test
– A small crystal of sodium chloride and two to three drops of glacial acetic acid are placed on a small piece of the suspected stain on a glass slide.A coverslip is applied and the acid is evaporated by heating over a small flame.It is examined under the microscope after cooling.Faint yellowish-red to brownish-black rhombic crystals of heamin or hematin chloride arranged single or in clusters are seen if blood is present.

Bubbles of gas are given by haematin crystals on addition of a drop of hydrogen peroxide.The reaction is negative if the stain is old, is washed or treated by chemicals,presence of too much salt,moisture in acid and over heating.

Spectroscopic examination
• It is the most delicate and reliable test for detecting the presence of blood in both recent and old stains.Less than 0.1 mg of blood is sufficient. • The blood is dissolved in water, normal sline or dilute ammonia and is placed in a small glass test tube which is then kept between the spectroscope and the source of the light.

• The extract of the blood must be dilute and if turbid it should be filtered. The solution of the blood has the property of absorbing some of the rays from the spectrum producing characteristic dark absorption bands which vary with the type of the blood pigment present. [see page 378]

4.The Blood is Human’s or not?— Species Identification
• Immunization-the provocation of immune • Method : 1. Ring Precipitation 2. Double agar diffusion test

Ring precipitation
• Principle : Blood serum contains proteins in colloidal suspension and when human serum is injected into an animal the animal becomes immunised against these proteins and antibodies develop in its blood.If human serum is then brought into contact with this animal serum the antibodies in the animal serum react with the proteins in the human serum and a visible precipitate forms.The antibodies causing this reaction are known as precipitin and the animal serum is known as antihuman precipitin test.

• A rabbit or a fowl is injected with human blood every third day for 3 to 5 injections.After this the animal is killed and the antiserum is collected.A suitable antiserum should react immediately or within a minute on the 1:1000 dilution • A white ring with well defined borders appears in case of positive reaction.The ring is mostly situated in the antiserum

• In case of negative reaction no ring appears • A positive reaction should begin in 10 minutes and should be read in half an hour. • For medicolegal purpose all the doubtful reactions are read as negative.

Double aggar diffusion test
• A piece of stained material or extract is placed in a central well cut in agar gel, while each of six well sourrounding this conains a drop of antiserum specific for the globulin of a particular species of animal.The advantage of this method is that extract can be tested against different antisera simultaneously.

5.Personal identification
• Who do the blood stain belong to? • By DNA typing, we can come to a conclusion that the blood stain belong to someone with a very high probability(>99.99%).

The examination of seminal fluid as trace elements

Civil importances
• • • • Compensation on the ground of acquired sterility Disputed paternity Artificial insemination Legitimacy Compensation on the ground of failure of vasectomy cases, leading to pregnancy of the wife • Divorce cases: non-ejaculation amounts to impotence

Criminal importances
• In relation to sex offence cases a) concerning commission of sex offence b) identification of the offender

Physical appearance
• When dried on cloth, the area appears slightly glistening, starchy hard in feeling, irregular in shape and distribution, and is whitish on dark clothes, and has fluorescence property when examined under ultraviolet light in a dark room

• Acid phosphatase is present in almost all body fluids. But its concentration in seminal fluid is maximum (about 20 to 400 times more than normal of about 20 units present in other body fluids). • this is a sensitive but not specific test. this test is a screening test, because many other body fluids also give positive reaction, However if the test is negative then presence of seminal fluid in the stain is excluded.

Confirmatory test
• Microscopic examination the dried seminal stain extract can be stained with some dye(methylene blue or eosin). Tail and the posterior third of the head of spermatozoa takes eosin or reddish stain and the anterior two third of the head of spermatozoa takes very light or faint, basic or blue stain.

Personal identification
• DNA typing

Confirmation Saliva stain 1. buccal squamous cells 2. amylase Vaginal fluid Vaginal epithelial cells stain Faecal stain Vegetable cells and muscle fibres Urine stain Urea and epithelial cell (from urethra or bladder)

Personal identification Nuclear DNA maker. Nuclear DNA maker Nuclear DNA maker Nuclear DNA maker

Parentage testing
• When the mother of a child names a person as to be the father of the child and the man denies this, it remains to be proved whether the putative father is the actual father of the child. Apart from morphological features, examination of genetic makers come for consideration to settle the dispute.

The basis of parentage testing
• 1. A gene will be present in a child, only if at least one of the parents has it; and that, • 2. If a locus in one parent is homozygous then its gene must appear in the child. • 3. A child must have a gene from each parent. • A child must, and can only, have a gene from each parent.

1 2 3 4 5 6 7 8 9 10 AF 12 13 14 L 111 M1 C1 AF2 M2 C2 AF3 M3 C3 L AF4 M4 C4





AF: alleged father; M: mother; C: child

If there are some loci that disobey the law, we can exclude the man as the father of the child. The only chance of exception of the theory is the chance of mutation which may occur in 0.1-0.2% new-borns. So at least 3 inconsistent loci are necessary for the exclusion conclusion.

Blood group

Red Cells Antigens



A Antigens

Anti-B antibodies


B Antigens

Anti-A antibodies


AB Antigens




Anti A and Anti B antibodies

Heredity of the blood group
Parents' Blood Types Possible Children Impossible Children A&A A&B A & AB A&O B&B B & AB B&O AB &AB AB & O O&O A, O A, B, AB, O A, B, AB A, O B, O A, B, AB B, O A, B, AB A, B O B, AB none O B, AB A, AB O A, AB O AB, O A, B, AB

Inheritance of MN types
Group of children parents Possible Impossible _______________________________________ M*M M*MN M*N N*N N*MN MN*MN M M,MN MN N N ,MN M,N,MN N,MN N M,N M, MN M None

Paternity Test Case
GM ABO LDLR GYPA HBGG D7S8 GC DQA1 CSF1PO TPOX THO1 D16S539 D7S820 D13S317 PI(总) M B BB BB BB BB AB 3,3 12,12 9,11 9,9 8,10 10,12 9,10 C B AB BB BB AB AB 1.2,3 10,12 11,11 6,9 9,10 11,12 9,10 AF O AB AB BB AA BB 1.2,1.3 10,12 8,11 6,6 9,11 11,12 8,9 PI 1.3632 2.3474 1.1382 1.8109 1.6428 1.5437 2.6838 2.0559 1.6567 8.8810 1.6119 1.6835 1.8031 6644.1011

• The paternity index (PI) compares the likelihood[probability] that a genetic marker (allele) that the alleged father (AF) passed to the child to the probability that a randomly selected unrelated man of similar ethnic background could pass the allele to the child.