Saturday, January 15, 2011


Determination of the type and characteristics of blood, blood testing, bloodstain examination, and preparation of testimony or presentations at trial are the main job functions of a forensic serologist, who also analyzes semen, saliva, other body fluids and may or may not be involved with DNA typing. It must be recognized, however, that in many crime labs, there may be no clear distinction between job title and job function. A particular laboratory may not have a serologist on staff, their functions being performed by a criminalist, a biochemist, a forensic biologist, or other technician. Such personnel would normally possess a Bachelor's or Master's degree, while a chief serologist would possess an M.D. or Ph.D. It's rare to find chief serologists, and the Bachelor's degree seems common. A few states have laws which make serological examinations admissible by statute without the necessity for testimony by an expert, the purpose of which is to insulate and protect their crime lab technicians. Other states rely upon their Chief Medical Examiner's office, forensic pathologists, or board-certified toxicologists. Professors of biochemistry, hematology, and immunology are often "borrowed" as experts by both prosecution and defense.

In certain specialized areas involving bloodstain examination (such as blood spatter analysis), courts will ordinarily qualify someone as an expert who has no formal education but specialized training and has conducted a sufficient number of examinations and accumulated enough reference patterns to be able to demonstrate the basis of their opinion. These kinds of experts are usually law enforcement personnel, and their testimony is most frequently found in those states which have modified Frye or embraced Coppolino. Further, blood and bloodstain evidence is such an integral part of most crime scenes that a police investigator/bloodstain specialist might be found, in some jurisdictions, testifying on the ultimate issue, even though this usurps the province of the jury. Federal Rule of Evidence 704 allows this to some degree. The Daubert impact has brought conditions more in line with Federal Rule of Evidence 702 than with a statistical showing of validity and reliability. Probability estimates are frequently used in blood testimony.

Blood is the most common, well-known, and perhaps most important evidence in the world of criminal justice today. There's no substitute for it, whether for medical or forensic purposes. Its presence always links suspect and victim to one another and the scene of violence. Bloodstain patterns tell a lot about position and movement during the crime, who struck whom first, in what manner, and how many times. This destroys most alibi and self-defense arguments for crime, and at the very least, trips most suspects up in their explanation of what happened. Over the years, criminals have tried many ingenious ways to hide, clean up, and remove blood evidence, but it's an area where criminal justice technology has always stayed one step ahead of them.

Blood is a slightly alkaline fluid made up of water, cells, enzymes, proteins, and inorganic substances that circulate throughout the vascular system carrying nourishment and transporting oxygen and waste. The most fluid portion of blood consists of plasma, which is mostly water, and serum, which is yellowish and contains white cells and platelets. The most non-fluid portion of blood consists of red cells which outnumber white cells by five hundred to one. While medical scientists are more interested in white cells, forensic scientists are more interested in red cells and secondly with serum. With serum, the analyst can determine the freshness of a blood sample because serum clots several minutes after exposure to air (a centrifuge is necessary to separate clotted material from the rest of serum). In serum are also found antibodies, which have important forensic implications. With red cells, the analyst looks for smaller substances residing on their surfaces, such as antigens, which have important forensic implications. One might even say that forensic serology is all about antigens and antibodies, but that is the domain of immunology.

In forensic law, blood has always been considered class evidence, but the potential exists for individualized blood typing, and even today, forensic serologists can provide testimony with some strong probability estimates linking a single individual, and that individual only, to a bloodstain. Consider that identical twins may have the same DNA profile but completely different antibody profiles, and you begin to see how promising the field of forensic serology really is.


The typing of blood, with what is now called the A-B-O system, was discovered in 1901. A few years later, starting around 1937, a series of antigen-antibody reactions were discovered in blood, the most common ones being ABH, MN, Rh, and Gm (over 100 antigens exist). Most people are only familiar with the Rh factor, which is technically the D antigen. There are more than 256 antigens, and 23 blood group systems based on association with these antigens.

There are a lot of components surrounding blood cells. Antigens are chemical structures attached to the surfaces of red blood cells. Antibodies are proteins floating in blood fluid (the serum, specifically, and platelets, associated with clotting), and exist because people have allergies or may have come in contact with a common disease (TB, smallpox, and hepatitis are common antibodies). Blood may also contain HIV antibodies, syphilis, and cholesterol. The most common problem (hematological condition) with blood is an iron deficiency. Iron is essential for the production of hemoglobin, the red pigment in cells, and iron also makes an excellent transport vehicle for nutrients. Anemia is a related condition involving a deficiency in the number of red blood cells.

A basic principle of serology is that for every antigen, there exists a specific antibody. In fact, ALL BLOOD GROUPS ARE DEFINED BY THE ANTIGENS ON THEIR RED BLOOD CELLS AND ANTIBODIES IN THEIR SERUM.

Blood type: Antigens on red cells: Antibodies in serum:
A A Anti-B
B B Anti-A
AB AB Neither anti-A or anti-B
O Neither A nor B Both anti-A and anti-B

For routine blood typing, all you need are two antiserums: anti-A and anti-B, both easily available commercially. By dripping a droplet of these antiserums in samples of blood, you see which samples maintain a normal appearance (at about 200x magnification) and which samples become clotted, or agglutinated. Blood of type A will be agglutinated by anti-A serum; blood of type B will be agglutinated by anti-B serum; AB blood by both; and O blood by neither. You are essentially determining blood type by injecting the worst possible poison into someone's blood sample to see what happens. Also, despite some racial and geographical variation, blood types are normally distributed in a population as follows:

43-45% 40-42% 10-12% 3-5%
O+ 39%
O- 6%
A+ 35%
A- 5%
B+ 8%
B- 2%
AB+ 4%
AB- 1%

The "O" type is most common among indigenous people (like Aborigines and Native Americans) and Latin Americans. The "A" type is most common among Caucasians and those of European descent. The "B" type is most common among African-Americans and certain Asians (e.g. Thai). The "AB" type is most common among the Japanese and certain Asians (e.g. Chinese). An interesting phenomenon is that Middle Easterners are somewhat likely to have nucleated red blood cells, whereas normally, red blood cells contain no nucleus. Men generally have more red blood cells than women. Red blood cells are originally formed from stem cells, and stem cells are found in bone marrow, the ribs, breastbone, pelvis, and vertebrae, but red cell production is controlled by a hormone released by the kidney, which in turn, instructs the bones to release more red blood cells. Rare blood types exist in addition to the basic ABO system.

A far more useful breakdown involves the Rh (Rhesus disease) factor. If a person has a positive Rh factor, this means that their blood contains a protein that is also found in Rhesus monkeys. Most people (about 85%) have a positive Rh factor, and doctors are trained to monitor closely any woman who is Rh negative and becomes pregnant. The Rh system is actually much more complicated than the ABO system because there are about 30 combinations possible, but for the sake of simplicity, Rh is usually expressed as either positive or negative. The Rh factor, like other antigens, is found on the covering of red blood cells. It's common for a forensic scientist to take the percentage distribution of the Rh component, which is expressed as plus or minus, to present some of the blood groups in terms of odds-ratios:

O+ 1 in 3 persons
O- 1 in 15 persons
A+ 1 in 3 persons
A- 1 in 16 persons
B+ 1 in 12 persons
B- 1 in 67 persons
AB+ 1 in 29 persons
AB- 1 in 167 persons

Subgrouping is also possible under the ABO system. Various extracts can be obtained from plants and seeds to create antiserums that clot type O blood, for example, somewhat selectively. Most major blood groups have at least two major subgroups; O1, O2, A1, A2, etc. The most commonly used types of antiserums used for this purpose are called lectins.

The possibility of individualized blood types is based on the typing of proteins and enzymes. Forensic serologists almost always do this level of typing. Blood proteins and enzymes have the characteristic of being polymorphisms or iso-enzymes, which means they exist in several forms and variants, so each one of them have subtypes. Most people are familiar with at least one common polymorphism in blood: Hb, which causes sickle-cell anemia. The following are some common polymorphisms:

PGM 2-1
erythrocyte acid phosphatase
esterase D
adenyl kinase
adenoisine deaminase
glutamic pyruvate transaminase
6-phosphogluconate dehydrogenase
glucose-6-phosphate dehydrogenase

Each of these protein and enzyme variants, as well as all blood subtypes, have known distributions in a population. It's therefore a simple matter to calculate probability estimates that border on individualized blood typing. (Let's do the math) Suppose you had a crime scene sample and a suspect which both were characterized by type A blood (42%), basic subtype A2 (25%), protein AK (15%) and enzyme PGM 2 (6%). The probability of finding two people in the population with this exact type would be less than 0.000945 (.42 x .25 x .15 x .06). The closer you come to producing a number out sixty decimal places, the more you've achieved saying there's no one else on Earth who could have committed the crime. Juries are usually impressed, however, by numbers out four, five or six decimal places, and the defense is put in the awful position of having to put a mathematician on the stand to lecture them about how many decimal places should be impressive.


The science of bloodstain analysis somewhat traditionally follows certain steps which serve to adequately describe the various tests conducted. Those steps are:

1. Is the sample blood?
2. Is the sample animal blood?
3. If animal blood, from what species?
4. If human blood, what type?
5. Can the sex, age, and race of the source of blood be determined?

To answer Question 1, forensic scientists use color or crystalline tests. It used to be that courts trusted police investigators who said they knew blood when they saw it, but that was before Miller v. Pate (1967) where someone got stumped on a cheap lawyer trick with red paint on clothes. The benzidine test was popular for awhile until it was discovered to be a known carcinogen, and was replaced by the Kastle-Meyer test, which used the chemical phenolphthalein. When it comes in contact with hemoglobin (and sometimes potato and horseradish), phenolphthalein releases peroxidase enzymes that cause a bright pink color to form. To detect invisible blood stains, the luminol test is used, which is a chemical sprayed on carpets and furniture which reveals a slight phosphorescent light in the dark where bloodstains (and certain other stains) are present. Long-dried blood has a tendency to crystallize, or can be made to crystallize with various saline-acid mixtures, and the names of various crystal tests are the Teichman test, the Takayama test, and Wagenhaar test. The generic term for any way of determining if something is blood or not is called a presumptive test.

To answer Questions 2 and 3, forensic scientists use antiserum or gel tests, and you may ask why it's important to test for animal blood. The answer is that any possibility of an injury to the household pet must be ruled out (or a fight between two pets, if pets are present). Pets normally spread human bloodstains all around the crime scene, but the pet can be a victim, perpetrator, or witness (by the transfer of animal DNA to the perpetrator). Veterinary forensics may be needed if pets are involved. Anyway, the standard test for telling if something is human or not is called the precipitin test, and is a technique that is based on injecting an animal (usually a rabbit) with human blood. The rabbit's body creates anti-human antibodies, which are then extracted from the rabbit's serum. If this antiserum is then placed on a sample from the crime scene, and creates clotting, you know the sample is human. The same procedure of creating and extracting antiserum can be extended to every known animal, but most labs buy the stuff commercially rather than keep a zoo on hand.

To answer Question 4, forensic scientists must first determine if they have an adequate and quality sample. If so, direct typing (as explained previously) using the A-B-O system is done. Indirect typing would have to be done on severely dried stains, and the most common technique is the absorption-elution test. It is done by adding compatible antiserum antibodies to a sample, then heating the sample to break the antibody-antigen bonds, then adding known red cells from standard blood groups to see what coagulates.

To answer Question 5, forensic scientists use various color and nitrate tests, as well as heredity principles to estimate things like age, sex, and race. No exact determinations are possible, but clotting and crystallization help estimate age, testosterone and chromosome testing help determine sex, and certain (controversial) racial genetic markers involving protein and enzyme tests helps determine race.

In addition, about 80% of the population are "secretors" which means that their other body fluids contain the same antigens, antibodies, and polymorphic enzymes as in their blood. In fact, the saliva and semen in such individuals have higher concentrations of A and B antigens than their blood. The forensic serologist often will want to analyze the stains of other body fluids.


Wet blood has more value than dried blood because more tests can be run. For example, alcohol and drug content can be determined from wet blood only. Blood begins to dry after 3-5 minutes of exposure to air. As it dries, it changes color towards brown and black. Blood at the crime scene can be in the form of pools, drops, smears, or crusts. Pools of blood obviously have more evidentiary value in obtaining a wet sample. Drops of blood tell the height and angle from which the blood fell. The forensic science of blood spatter analysis says that blood which fell perpendicular to the floor from a distance of 0-2 feet would make a circular drop with slightly frayed edges. Drops from a higher distance would have more pronounced tendrils fraying off the edges (a sunburst pattern). A blood smear on the wall or floor tells the direction of force of the blow. The direction of force is always in the direction towards the tail, or smaller end, of the smear, or splatter. In other words, the largest area of the smear is the point of origin (a wave cast-off pattern). Blood crusts need to be tested with crystalline methods to make sure it's blood.

Refrigerated red blood cells have a shelf life of about 42 days, and the serum containing white blood cells can be refrigerated much longer, almost up to a year. DNA can be extracted from blood (if white blood cells which always contain a nucleus are present), and also from sperm, bone marrow, tooth pulp, and hair roots. Blood, however, is commonly used in DNA testing, as per the following steps:

1. Blood samples are collected from the victim, defendant, and crime scene
2. White blood cells are separated from red blood cells
3. DNA is extracted from the nuclei of white blood cells
4. A restrictive enzyme is used to cut fragments of the DNA strand
5. DNA fragments are put into a bed of gel with electrodes at either end
6. Electric current sorts DNA fragments by length
7. An absorbent blotter soaks up the imprint; it is radioactively treated, and an X-ray photograph (called an autoradiograph) is produced

Case study

When police have a strong suspect in a murder case, the temptation is to leave it at that, to close down the search for a killer. But a few blood samples submitted to tests in the forensic laboratory can change the entire case!

Good blood cannot lie, they say. Nor can bad. As the distinguished forensic expert Alixtair R. Brownlie (Solicitor Supreme Courts, Edinburgh. Scotland) put it to Britain's Forensic Science Society: "Since Cain slew Abel, spilt blood had borne its mute testimony in crimes of violence. Stains of blood and body fluids still play an important part in crime detection, a lesser but increasing part in the proof of guilt. And not only the nature and grouping of stains, but their position at the scene of the crime can be revolving and is now recognised as a vital piece of evidence in itself.

The investigation of blood at a crime scene can be broadly divided into a biological approach (serology) and a physics approach (blood splatter or bloodstain pattern interpretation). This fact file will concentrate on the serological approach to blood evidence. Another fact file will cover the bloodstain pattern interpretation.

Blood is not the only body product, which can be of use to the forensic blood grouper. The word serology comes from the ancient Sanskrit sara, meaning, "to flow". Today it is known that every fluid, which flows in the human body, can be identified: sometimes to prove the guilt of a suspected person, but also very often to protect the innocent. .

Essentially, forensic serology is based upon facts known vaguely since the dawn of time, and with much more certainty since in1628 the English physician William Harvey discovered the circulation of blood. Christopher Wren is said to have experimented with transfusion, and in his diary Samuel Pepys recorded that a donor was paid a sum of 20 shillings (about $500 in 1974 money), as well as speculating what would happened "were the blood of a Quaker to be let into an Archbishop". For centuries the English aristocracy were genuinely believed to be born with blue blood, and boasts such as "the blood of an Englishman" were taken seriously.

Then, in 1930, the Viennese doctor Karl Landsteiner received a Nobel Prize award for his research into serology. He had announced to the scientific world that all human blood could be grouped into four main types. His work stimulated other biologists. Today for convenience the groups are known as O, A, B and AB.

Expert Evidence

While it should be remembered that it is never possible to say "this bloodstain originated from this person"; nevertheless it may be possible to conclude, "this bloodstain cannot have originated from that person". A defence case may depend on this crucial fact. One striking example came to light early in September 1961 in England, when a 24 year old army private at Aldershot was cleared of sexual attack on a 38 year old mother.

"I can't remember exactly what happened," the woman said to the police "He jumped on me and got hold of my shoulders. I screamed as hard as I could. Then somehow I found I was at the bottom of a steep bank, and my little daughter was crying. The man had pulled off my blouse, but I gave up the struggle because he twice threatened to hurt my child .."

False Identification

The doctor who examined the woman later confirmed there had been an attack. A solider was picked out at an identification parade, and charged with rape. Bryan Culliford, from the New Scotland Yard Laboratory, demonstrated that tests proved the suspect was in Group B, while the stains on the unfortunate woman were Group A. "We find there is no case to answer" announced the chairman of the court.

In her distressed state, the woman had picked out the wrong man at the identification parade. But for serology and its forensic application an innocent man could have been sent to jail.

Blood continues to play an important part in forensic investigations, and the discovery of new antibodies has enabled blood grouping techniques to be further refined. For example the Kell antigen is virtually confined to the white population, whereas the Duffy antigen is completely absent. Thus, blood grouping characteristics can be used to give an indication of race, and help to pinpoint the origin of bloodstains.

Forensic laboratories have researched sophisticated techniques for analysing protein in blood, and have been able to produce blood profiles with the prospect of establishing unique blood "fingerprints". While this remains for the moment a serologist's dream, blood continues to give up its secrets, and has described it as 'a treasure trove of hidden clues'.

The first task in examining suspicious stains is to determine whether they are blood, and if so, are they human? Once this is established stains are examined for age, sex and blood group. The shape and pattern of liquid blood-splashes can help in reconstructing the murder; bloody fingerprints and palm-prints tell their own story; dried blood on a suspect's clothing can be related to the victim, the crime scene and the murder weapon; blood and tissue forced under the fingernails of the victim during a violent struggle can be linked to the assailant.

Thus a single blood-trace can provide a wealth of information, and analytical techniques are improving all the time. For example, traces of drugs found in a bloodstain indicate medical treatment which a person might be receiving. While such procedures improve the scope of detection, it is not yet possible to identify an individual by his blood as it is by his fingerprints. Nevertheless, forensic serology, which in addition to blood deals with other body fluids such as saliva and semen, is important not only for narrowing suspicion on the guilty but also in showing a suspect's innocence. As in many other aspects of forensic investigation, bloodstains are taken into account with a variety of other evidence to build up a pattern of crime.

A number of substances such as fruit-stains or dye-stuff may soil clothing and take on the appearance of bloodstains. The benzidine test - used for many years to confirm the presence of blood - has been discontinued because the reagent is carcinogenic. It has largely been replaced by the Kastle-Meyer test, using a solution of phenolphthalein which turns pink in contact with even small traces of blood. The test works by detecting the presence of the enzyme peroxides in the blood. However, as this substance is also present in other biological materials, the Kastle-Meyer test is regarded as a screening procedure. It is highly sensitive, and positive reaction is judged presumptive of blood, and further confirmatory tests are carried out. These are usually chemical and microscopically procedures to identify blood by its pigments and cellular structures.

Once a stain has been confirmed as blood it has to be determined whether it is human or animal. The precipitin test is used for this purpose. Blood of every animal species contains different proteins, and blood from one species will not accept proteins from a different species. Blood develops antibodies as a protective measure against disease and foreign matter to render them harmless. The serum containing antibodies produced by this reaction provides immunity from disease.

This principle is used to test whether blood-stains are human or not. Serum for the precipitin test is obtained from rabbits which have produced antibodies to destroy a small quantity of human blood injected into them. A drop of this anti-human serum is added to suspect blood, which will precipitate its protein if it is of human origin. Police laboratories hold anti-sera for most common animals, thus allowing the crime investigator to confirm or disprove statements made by the suspects about he origin of suspicious bloodstains. The precipitin test is sensitive, and will work on small traces of blood. The test is also known as the Uhlenbuth test after the German scientist who developed it in 1901.

The colour of dried blood changes in time from red to brown, and the peroxidise test takes longer to develop with an old stain. An experienced observer considering these factors might be able to give an opinion as to the age of a particular stain, but it is now possible to measure colour-change scientifically. Spectrophotometric analysis of bloodstains allows them to be aged within the range of one day to three weeks.

In 1949 two British scientists observed that the nuclei in the cells of female tissues usually contained a distinctive drumstick - like structure which was rare in males. This structure called a Barr bodies after one of its discoveries, is most noticeable in white blood cells and in the epithelial cells lining the mouth. Barr bodies are associated with the differences in chromosomes between males and females, and their appearance in blood of unknown origin is a basis for identifying it as from a female.

Determination of the blood group characteristics of stains found on clothing or a suspected murder weapon is another powerful link in the chain of evidence that can be built up in a case of violent death. Blood grouping is a developing science in its own right, and while it cannot provide information as certain as a fingerprint, it can provide circumstantial evidence establishing contact between a suspect and the victim.

Every person's blood falls into one of the four international blood groups identified in 1900 by Dr Karl Landsteiner. The ABO blood grouping system is a function of the red blood cells, and the presence in them of a substance known as agglutinogen. A Group contains A agglutinogen B Group has B agglutinogen, AB Group contains both and O Group has neither. (What are anti-body reactions?) These factors are found in specific proportions among different populations.


O positive 40%
O negative 9%
A positive 31%
A negative 7%
B positive 8%
B negative 2%
AB positive 2%
AB negative 1%

What about other ethnic groups?

In 1927 Dr Landsteiner and a fellow-worker discovered further factors which occurred separately in human blood and were distributed in specific proportions among the population. These are the M. N. and MN factors, to which was added the P factor and in 1940 the Rhesus factor. The knowledge that each person's ABO and MN blood group characteristics are inherited and fixed for life has made the examination of blood an important part of crime investigation. It is possible to place an individual in one of 288 different blood groupings, but forensic serologists are not able to say that a particular blood trace originated in a particular individual. The value of blood grouping procedures in crime work is that many potential suspects can be eliminated from an inquiry, thereby allowing the investigation to be narrowed down. About 80 per cent of the population are secretors which means that their blood cells are present in such body fluids as semen and saliva. It is possible, therefore, to determine blood groupings by examining these fluids.

Blood test

In criminology scientists do concern themselves with medical matters such as agglutination, but primarily the vital question involves whether or not a sample is blood. A minute sample in the laboratory is extracted from the stained material kept in a saline solution, and a tiny drop of the extract is mixed with a solution containing phenolphthalein and potassium hydroxide, powdered zinc and hydrogen peroxide. If this test is negative (no change), the sample cannot be blood. If the mixture shows a clear pink colour, it is blood.

Biologists sometimes use a different test, in which glacial acid is added to a solution of hydrogen peroxide and benzidine - a drop of this being added to the test sample, which immediately turns a deep blue if there is blood present. The next step is to use an antiserum prepared in an animal, which will react specifically with human blood, thus demonstrating whether the sample is of human origin.

"The blood of an Englishman" is not a subject over which forensic serologists wax racialist, because crime is international. The frequencies of the various genes within different blood group systems may, however, vary from race to race and could possibly provide important evidence. Blood group systems in general have acquired names such as Kidd, Duffy and Kell after the patients in whom the antibodies were first discovered, and all of them, of course, allow scientists to narrow down the field.

Summarizing all the international work of forensic serologist, the late Dr F.I.N. Dunsford, Ph.D. of Britain's National Blood Transfusion Service, stressed that, in crime detection, the "usefulness" of a blood group system is the measure of its efficiency - differentiating the red cells of one person from those of another.

From his tests, for example, it is known that the Rhesus antigen V is present in fewer than 0.5 per cent of white people, but present in 40 per cent of West African Negroes. The chromosomes (the rod like structures which show as pairs in every developed cell) known as cDe are also more common among Negroes than whites.

The Duffy phenotype Fy is always completely absent from whites, but present in 90 per cent of West Africans. Kell antigen is virtually confined to white races, while Diego positives are virtually absent from whites, yet present in Caribe Indians, Japanese, and Chinese.

At the extreme of the blood group sis a certain LU (a _ b__) factor, which many serologists believe to be so rare that an estimated total of only eight people among the world's 3200 million plus can have it. One of the eight, a Sheffield ( England ) woman, had three pints of the rare blood flown to her in a British hospital from an American donor.

More on blood type systems.

However, researchers maybe only on the threshold of discoveries in investigation of body fluids. It is now nearly 75 years since serologists put blood samples under the microscope and found the elements which are freely suspended in the plasma - essentially the erythrocytes (red corpuscles), leukocytes (white corpuscles), and the blood platelets (egg shaped and circular bodies suspended in the straw plasma more commonly known as the "serum").






Compression type tool marks are produced when a tool strikes the surface with a pressure. Striated type tool marks are produced when a tool scrapes the surface. Repetitive type marks are produced when a tool strikes the surface a number of times.

The features of the tool marks can be divided into three parts as class characteristics, sub class characteristics and individual characteristics.

1. Class Characteristics: These are the features produced by similar type tools.

2. Sub class characteristics: These are the features produced by tools of one type manufactured on the same machine.

3. Individual characteristics: Some individual characteristics are developed by a tool while in use due to wear and tear. No two tools can produce identical random individual marks.

Examination of tool mark

Whenever a tool mark is found at the scene of crime the following procedure should be adopted for its collections and examination.

1 Photography: Tool marks should be photographed. At least two photographs should be taken, one showing the background with the impression and another a close up of the impression. Scale should always be kept at the same level of the tool marks while taking the photograph. Camera should be kept perpendicular to the surface of the tool marks. More details are obtained by using oblique illumination.

2 Tracing: Tool marks can be traced on a tracing paper. This helps in comparing the class characteristics.

3 Lifting of tool marks: A cast or mould is simply a reverse or negative three-dimensional image of an impression. The following materials are generally used for making a mould of a tool mark.

Plastic/rubber: This is a material for lifting fine details of a tool mark. A lump of material is softened by pressing in fingers and then applied on the surface. It is pressed carefully as it is likely to be disturbed by out side pressure.

Dental casting material: This also gives fine details of tool marks.

4 Plaster of Paris: This material is used when the impression is of large size. Plaster of Paris is partly hydrated calcium sulphate (CaSO4)2 1/2 H2O. First on the surface of tool marks talcum powder is sprayed. This helps in removing the cast without disturbance. Then solution of plaster of Paris is put on the surface. This is allowed to dry and the cast removed carefully. For preparing plaster of Paris solution, water is taken in a container and plaster of Paris added until water does not absorb any more plaster of Paris. 7 parts of plaster of Paris are generally put in 4 parts of water. To increase rigidity of surface details a thin layer of shellac dissolved in alcohol is sprayed on the surface by means of sprayer of the type used for spraying insecticides. The shellac is carefully sprayed from a distance of several feet so that air pressure does not disturb the details. The plaster is then poured in. When the caste has hardened the shellac is peeled off. The talcum powder permits the peeling off the shellac without affecting the cast.

5 Metal casting: The mould of tool mark can also be made by metal casting. The suitable metal for this purpose is known as woods metal. This is a metal of low melting point (71oC), which is suitable for casting tool marks. Its composition by weight is Bismuth 50%, Lead 25%, Tin 12.5% and Cadmium 12.5%.

6 Cellulose acetate: Tool marks from stone, concrete, wood, etc. can be lifted by cellulose acetate dissolved in acetone. A layer of 1/16 inch is made. However the cellulose acetate cast should be photographed, with scale, immediately otherwise the layer is likely to shrink.

Examination of foreign material/materials on Tool

The suspected tool is examined for presence of all traces of metal or wood and foreign particles sticking to the tool/blade using the stereo microscope. The physical and chemical characteristics of these particles should be compared with the material of the surface on which the tool mark was made.

Taking test impression

The material on which the test impression is to be made must be chosen carefully. It must be soft enough so that the tool edge will not be altered. When the original mark is in wood, paint or soft metal, the standard should be made with a similar material. Several test impressions should be made at various angles with the tool.

When the original mark is on hard metal such as steel or brass, it is desirable to use the same material for the standard. Sheet lead, tin or aluminium may be employed successfully.

Comparison of tool marks

The tool mark is negative replica of the tool and mould is negative replica of the tool mark. A tool mark must be compared with tool mark, mould with mould and photograph with photograph having same magnification. The comparison is best done by using comparison microscope and with oblique illumination. Magnification of test and crime sample should be identical.

Specific problems:

1- To determine whether a lock is forced open or not

The key manufactured for a specific lock always plays through a prescribed path inside the lever mechanism of the lock with out causing any scratch on the surface of the levers. On the other hand, if a duplicate key, foreign tool, etc, are used to open a lock, then several fresh scratches can be observed on the levers and internal surfaces of the questioned lock. If the lock was opened by applying heavy mechanical thrust, tool marks can be found on the body of the lock.

The lock is examined under a stereo microscope to locate the point of disturbance and tool marks on the body of the lock Then the questioned lock is cut open in such a way so that the lever mechanism remain intact. The lever mechanism is to be observed under a stereomicroscope in oblique illumination for any tool marks or other disturbances. Usually, the recent marks and scratches have bright metallic lustre. If the surface of the lock bears any tool marks or deformation caused due to hammering with tools, then the entire defect and abnormality are to be matched with respect to its original position of locking

2- Tool marks in the case of cutting of electric wires/cables

Crimes involving the use of wires are not infrequent. The clipping of cables and wires is found in wire and transformer theft case sabotage cases etc. The cut wire is used as evidence material. If the wire cutters are also seized, the tests impression/exemplars are produced and compared with those on the evidence cut wires.

3- Tip of the broken tool

If a broken piece of tool tip is found embedded in the surface, the physical matching of the broken part with the suspected tool gives a definite proof of the tool used in the crime. The broken part may also be found fallen on the ground at the scene of crime.

4- Staple marks

Use of staple is increasing day-by-day due to its low cost and convenience. Two types of problems are encountered in the staple cases. One is source identification and second is to link the stapler with the staple. For source identification the marks of manufacturing of the staple can be compared under microscope. Material of the staple can be compared with elemental analysis techniques. To link the stapler with the marks on the pin, the test marks are made with the questioned stapler and compared with the marks present on the crime pin, by conventional tool marks comparison techniques.

Methods Used to Obliterate Identification Marks

1 Filing or grinding- The original number would have been filed away or ground with a power grinder followed by polishing and then over stamping with a new number.

2 Peening- This involves hammering the surface with a round punch to hide the number.

3 Over stamping- Here a new number is simply stamped over the old. For numbers with curved surfaces i.e. 2,3,5,6,9 and 0, the stamp 8 is the one most often chosen. For numbers with straight surfaces i.e. 1 and 7, the stamp 4 is the obvious choice. Serial numbers with a preponderance of, 8 or 4 numbers should be treated with suspicion.

4 Centre punching- The surface bearing number is obliterated with a pointed punch.

5 Substitution- Substitution of an iron plate with a new number over the original surface by pasting or welding.

6 Drilling- It removes the number and the surrounding metal with a drill. The cavity is usually filled up with either lead solder or welding material.

7 Welding- Heating the surface with either an oxy-acetylene welder or an arc -welder until the metal flows.

8 Occasionally an original finish would be given to a previous obliterated number surface

Chemical etching method for restoration

The chemical etching method is the simplest and most effective method for restoring obliterated numbers. It is simple to apply and it requires no expensive equipment. It works well on any size or type of object.

The techniques involved require considerable skill and great patience. The materials used are potentially dangerous and should be used with full awareness of health and safety requirements. They should only be used in areas specially set aside for this purpose. Rubber gloves will protect the hands from corrosive acid and when using volatile liquids work in the open air away from any seat of naked flame, if laboratory conditions are not available.

In case of motor vehicles remove the engine from the vehicle if necessary for ease of access to the engine number surface, and for better quality of photographs of the restoration process.

1 Preliminary Examination

Examine the metal surface after cleaning oil and dirt away, using acetone. Examine the surface with a hand magnifier and see whether any erasure has taken place at all. Look for any disturbance in the pattern at the background. This pattern will be present either in the form of milled marks caused by grinding the surface before stamping the serial numbers, or cast marks produced during the manufacturing process. If it is disturbed, suspect erasure. Even if no erasure is noticed, remove the paint over a wide area around the surface to check whether the portion carrying the chassis number was removed by cutting and substitute for it by welding or pasting a metal plate with a new number4 . Remove the pasted plate if any, from the original surface by using trifluoroethanol, after recording the original appearance. Remove the welded plate too. Examine the lower surface for any obliterated marks. Note how the mark has been erased and whether it has been repaired after erasure. See if any digits or parts of digits are visible. Note these down. Examine the surface by carefully adjusted illumination preferably oblique lighting to see the erased number.

For photography, use a single light for striking the surface bearing the serial number at a low angle. Take several exposures, the light being moved in each instant to strike the surface from a different position. Use process film. This method assists to bringing out the faint serial numbers.

Identify the type of metal from which the object is made. This is necessary to choose the appropriate chemical etchants.

2 Preparation of surface

(i) Clean the surface using preferably benzene or acetone to free it from grease or paint. Solvents such as gasoline, commercial paint remover or 50/50 mixture of acetone and chloroform may also be used. To assist the solvent a soft toothbrush should be used to dislodge deposits from the stamped surface.

(ii) Hand polish the area to a smooth, mirror like finish or a reasonably smooth surface with emery cloth, or other fine abrasive. Emery paper with coarse grade is used by first removing all scratches, and other gross marks with fine emery. Do not remove more metal than is necessary. Leave deep scratches. Examine the surface during polishing, as digits are sometimes revealed during the process.

(iii) Clean again the area with a solvent, such as acetone or other grease solvent. Do not touch the area with bare fingers because fingerprints can interfere with the reaction.

(iv) Heating- Treat the area with a blow lamp or Bunsen burner. Care should be taken not to over heat. If the metal is heated to red hot, the temperature is sufficiently high to soften the metal and, on cooling, the metal becomes homogeneous and can no longer be differentiated. A useful guide is to heat the metal surface until it is just too hot to touch. The metal should be allowed to cool before etching begins 1, 2.

(v) Photograph the entire item, and record details of the obliterated area by close-up photography. Use 35 mm camera with a macro lens and extension and a fast film, 400, ISO.

Etching procedures on different surfaces

1 Steel surfaces (chassis and engine of cars, guns, gas cylinders etc.)

(i) The etching reagents are in two solutions.

Solution 1 (Fry's reagent)

Crystalline cupric chloride 90gms.

Concentrated hydrochloric acid 120ml.

Water 100ml.

Solution 2

15 per cent Nitric acid.

Apply the Solution 1 by swabbing the surface for approximately 2-3 minutes with cotton wool dipped in the reagent. Look for any digits revealed and record these. Clean the surface with acetone (not water). Examine again.

Apply the Solution 2 by swabbing the surface for approximately 1 minute with cotton wool dipped in this solution. Look for any digits revealed and record these.

Continue etching alternatively with Fry's and 15% nitric acid etching reagents until the complete number is visible. This may take up to 2 or 3 hours. A consistent check should be made on the appearance of the mark since faint marks appear and then disappear again before the mark is completely etched.

(ii) Alternate Solution

Solution 1

Hydrochloric acid 80ml.

Water 60 ml.

Copper Chloride 12.9 gms.

Alcohol 50 ml.

Solution 2

15 per cent Nitric acid.

The method is similar to the previous one. Apply with a swab a little of the acid copper chloride solution and continue to apply for 60 seconds. Dry with cotton wool and then apply the nitric acid for a similar period.

This alternate swabbing with solutions (1) and (2) is continued until the number is developed. Time, 5 minutes to as long as 1 hour.

(iii) Ferric chloride etching reagent can also be successfully used on steel surface.


1. If etching results in areas of dark contrast surrounded by metal with normally etched appearance, this indicates that the mark has been torched with a welding torch, and further efforts to restore the mark are a waste of time.

2. When nitric acid reagent is applied after the Fry's reagent, copper is sometimes deposited on the surface. This does not matter. It can usually be removed by rubbing vigorously with the nitric acid swab, or alternatively, it will disappear when Fry's reagent is applied again.

2 Copper, Brass, German Silver and other Copper Alloys

These metals react to:

Ferric Chloride 19 gms.

Hydrochloric acid 6 ml.

Water 100 ml.

Apply the reagent as a swab until the number appears. When the reagent is slow to develop the erased mark, form a wall of plasticine around the erased mark. Use the solution as a bath. Let the reagent remain on the metal for 24 hours. Some workers have found that 20 gms. Ammonium persulphate made up to 100cc., with water gives the best result for brass and copper.

3 Stainless Steel

Swab either with dilute sulphuric acid or 10 per cent solution of hydrochloric acid in alcohol.

4 Lead (Motor car batteries etc.)

Glacial acetic acid 3 parts

Hydrogen peroxide

This solution has yielded excellent results. Time 10-30 minutes. When the erased number ppears, clean the metal in concentrated nitric acid.

5 Zinc Alloys

(i) Sodium hydroxide 10 per cent solution in water.

Development is slow. So, use the plasticine bath method. Time 10-16 hours.

(ii) Alternate solution

Chromic acid 20gms.

Na2 SO4 1.5 gms

Water (or) 100 ml.

Nitric acid 25%

6 Cast Iron and Cast Steel

Apply constantly a 10 per cent solution of sulphuric acid plus potassium dichromate. Action will be slow. Apply constantly the reagent. It may even be necessary to build a wall of plasticine around the number and fill the hollow with the reagent. Remove the solution at intervals, examine the surface, replace fresh solution. Over development results in the obliteration of the restoration.

7 Aluminium Alloys (Engine surface of the motor bikes, auto-rickshaw engine, vehicle identification plates etc.)

(i) The best reagent is Villela's solution.

Glycerin 30 ml.

Hydrofluoric acid 20ml.

Nitric acid 10 ml.

Apply the reagent as a swab until the number appears. Use the reagent with caution as it attacks the fingers vigorously. Time 5-15 minutes.

(ii) An alternative and safer formula is Hume Rothery solution and is made up as follows:

Copper chloride 200 gms.

Hydrochloric acid 5 ml.

Water 1000 ml.

As soon as this solution is applied a copper deposit is formed. Remove this copper deposit by alternative swabbing with Hume Rothery reagent and distilled water, until the number appears. Time 5 minutes to 1 Hour.

(iii) FBI Laboratory recommends the following for etching aluminium surfaces

Apply the ferric chloride reagent to the surface to be etched. Several hours of application may be necessary to reveal the erased mark. Various methods of application can be used to avoid constant swabbing for hours, such as:

Soak a large piece of cotton wool in the reagent and it will stick to the metal surface by surface tension. The cotton wool must be removed and resoaked in the reagent at 20 minutes intervals, and the effect closely noted. If the surface to be etched can be made to be horizontal, a puddle of reagent can be left over the area, and replaced when it has lost its colour. Do not, however, build up a plasticine bath around the area as the etching reagent attacks plasticine.

Inspect the surface periodically. When the number is visible, clean the surface with acetone and dry it.

8 Tin

Hydrochloric acid 10 per cent solution.

Alternate swabbing and washing. Time 10-20 minutes.

(i) German Silver

Ferric chloride 25%

Hydrochloric acid(conc.) 25%

Water 50%

Swab the area. Time 10-20 minutes.

(j) Silver

Concentrated nitric acid

Immerse the specimen for few seconds. After each dipping wash with running water.

9 Gold and Platinum

Nitric acid 1pt.

Hydrochloric acid 5pts.

Distilled water 6 pts.

Carry out etching at 15oC. Time 1 hour or longer.

10 Wood (punched marks)

Many wooden articles are stamped with identifying markings which may be erased by cutting, filing or the use of abrasive. Provided the area beneath the exposure has been disturbed by the original punching, legible results may be obtained. Play a jet of steam onto the erased surface. The steam softens the wood and causes the fibres which were bent during punching the number to spring back and the broken fibres to swell. Where the mark has been, the wood now projects above the surface and the mark can be deciphered. Pretreat with a solution of caustic soda or potash for hard woods such as are employed for tool handle. This softens the wood and accelerates the treatment.

11 Leather

(i) Try all the methods of development given below, as the treatment of leather is empirical. Clean the surface with cotton-wool swab soaked in chloroform, and examine under ultraviolet light. This works admirably when the embossed letters have been filled with an oil paint. The oils are brought to the surface by the chloroform and outline the letters as a bright fluorescence.

(ii) Swab the surface with cotton wool soaked in 2N caustic soda solutions. The loose disturbed surface absorbs the solution, softens and darkens. The compressed leather, which has been embossed, does not absorb the solution so quickly. This differential absorption enables the erased mark to be deciphered.

(iii) When the carbon ink has been used on the embossed number and the same has been soaked into the surface.

Clean the surface as above and take an infrared photograph. When the ink has been a carbon ink and has soaked into the surface, cleaning in this way gives a surface which photographs as white under infra-red while the traces of carbon ink are photographed intensely black.

12 Rubber

Some rubber articles are stamped, and when stamping is erased the suspect area can be treated by lightly swabbing with petrol or even on occasions carbon-disulphide. This latter solution acts upon rubber very rapidly and should be used with caution.

13 Polymers

(i) It is not possible to make use of alteration of structure in the case of polymers. These materials have no organized macro structure and the die does not, therefore, produce alterations of structure capable of being treated with reagents. Numbers or other markings in a polymeric material are presently produced using a heated stamp. Laser drilling is also used to produce an identification mark.

Polish the surface as before and then flood the surface with a deeply colored solution such as a dye solution. When excess solution is removed, the dye soaked into the crevices of the mark is plainly visible and the mark can be deciphered.

(ii) Conduct experiments using swelling techniques with various solvents, relief polishing and heat-treatment. Follow the table below.

Polymer type


Polyethylene (high density) (PE-HD)

Heat (100oC, 5 min.), swelling with ethanol, light petroleum (40-60)

Styrene acrylonitrile (SAN)

Polishing heat (140oC, 10 min.), swelling with light petroleum (40-60)

Acrylonitrile butadiene styrene (ABS)

Polishing heat (100oC, 10 min.), swelling with light petroleum (40-60)

Polyamide (PA 6)

Polishing heat (150oC, 5 min.), swelling with light petroleum (40-60)

Polyoxymethylene (POM)

Polishing heat (150oC, 30 min.), swelling with light petroleum (80 oC)

Polybutylene terephthalate (PBTP)

Polishing heat (200 oC, 10 minutes)

Polycarbonate (PC-GF)

Swelling with ethanol, ethyl ether, heat (120 oC, 5 min.)

Epoxy resin (EP)

Swelling with acetone, formic acid, ethyl acetate, light petroleum (40-60)

Visualization and photography of the restored marks

Restored marks are to be distinct enough to be visualised and photographed, as far as possible. Better visualisation and photography can often be obtained if the etched surface is moistened slightly with etchants. There should be sufficient lighting to view the restored number because of the difficulty to view properly the number especially on the engine and the chassis of the motor vehicle. Portable beam lighting can be positioned to produce low angle illumination across the surface area for viewing.

However, for photography a powerful light source adjusted suitably for catching their images on the camera screen should be available. Flexible powerful fibre optics illuminators are excellently suited for this purpose. A 35 mm camera screwed on a tripod for time exposure will be very useful. A macro lens with extension rings would enable the camera to keep a close distance from the number area and ensure a magnified image on the film. Use a fast film 400 ISO. A photograph with a contrast film (100 ISO) should also be made. Process the negatives in the usual manner. Print on a normal grade paper for normal contrast.

Methods for restoration of obliterated engraved marks

1 Scope

Identification of articles of plate and jewellery, and common articles like stainless steel and tool steels by restoring the obliterated engraving identification marks.

2 Engraved Marks

Engraved marks are made on plate and jewellery using an engraving tool and engraved marks on hard metals like stainless steel and tool steels are produced by an electric engraver. All engraved marks are made by removing the metal with the tool. The underlying metal is not seriously disturbed, and no routine guaranteed method of restoration is possible.

Methods of Restoration

(a) Use (HCL) etching methods for restoring engravings on plated articles. Use acidic ferric chloride on chromium plated on brass plate. Use Fry's reagent for chromium-plated on mild steel plate. Choose an etching solution which preferentially etches the two metals. (b) In case of electrical engraving, the instrument heats the metal at the point of engraving, and partially fuses it. If the number has been filed out, polish the surface carefully. The remains of the fused areas reflect light slightly different from the rest of the metal. Therefore, throw a beam of light onto the polished surface and view through the correct angle. The erased number will be seen as a dark shadow on the bright background

1 Preservation of the restored punched and engraved marks

1. If a mark is successfully restored, it is important that the number is recorded and, as far as possible, preserved. In an attempt to preserve the mark, the area should be washed with dilute ammonia solution to neutralize the acid (if the surface is acid-etched), cleaned thoroughly with acetone and dried. Clear lacquer should then be thickly sprayed over the mark.

2. It is also important to clean up around the mark. Etching reagents are often splashed or dropped over surrounding parts of the article being examined, and if not cleaned off, corrosion can result.

3. Remember that different parts of marks will appear and disappear as restoration proceeds. A series of records may be required.

Methods for restoration of obliterated painted numbers and other marks


Identification of motor vehicle, registration number plate etc., by restoring the obliterated original painted number, and sticker numbers.

Methods of obliteration

Some number plates consisted of letters and figures painted onto the metal plate. Obliteration normally consisted in painting over the surface with black or other paint and inserting the false numbers. In a similar manner some floral and ornamental designs, registration numbers and some other identifying features all originally made on the painted motor vehicle surface are obliterated with new designs and numbers.

Methods of restoration

1- Take photographs the suspected obliterated surfaces before treating it in any manner. Examine the number plate and the vehicle surface at different illuminations, especially the oblique one. The obliterated marks may sometimes be visible.

2- Wash away the fresh paint and the fresh number with chloroform or dioxan. Remember that this action has its dangers because the underlying paint and the marks tend to wash away along with the top layer. Try suitable solvents mixtures of solvents at some other place and thus choose the suitable solvent or mixture of solvents for application in the obliterated surface

3- Apply the chosen solvents in cotton swabs over the paint surface suspected of being obliterated. Wait till the top layer just swells. Remove this layer carefully by gentle rubbing with cotton. Look for the obliterated marks and record them by photography and detailed notes. Take care that the underlayer is not washed away and removed along with top layer. Use the above procedure to discover more than one obliterated marks underneath

4- X-ray shadowgraph- If the article is portable try this method. Best results are possible when the underlying layers are painted with a paint containing heavy elements, such as lead, while the top paint is free from such elements. With the correct exposure the outlines of obliterated marks can be deciphered.

5- In the case of number plates where original sticker number is removed or replaced, try specular reflection. Throw an even light on the plate from an angle and photograph the plate from above. The brightly polished areas, which have been protected by the lettering, reflect the light specularly away from the camera. The weathered areas diffuse the light to some extent and some of this diffused light enters into the camera. As a result, the lettering appears black on a light background

6- Expose the plate to ultraviolet radiations. If the area fluoresces in ultraviolet light record the appearance by photography. Photograph following the techniques of UV fluorescence photography.

7- A photographic records of the restored marks should be made.