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:|
|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:
|O+ 39% |
|A+ 35% |
|B+ 8% |
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
glutamic pyruvate transaminase
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.
THE CRIME SCENE AND BLOOD
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
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.
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 .."
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.
FREQUENCY OF BLOOD GROUPS IN AUSTRALIAN POPULATION
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.
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").