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Forensic serology is the application of the study of blood, semen, saliva and
other body fluids to legal matters. The field generally is comprised of the
detection of enzymes and antigens, as in the identification of seminal stains
or blood typing (ABO and secretor status) and DNA typing (by PCR or RFLP

An interesting application of combined forensic toxicology and forensic

serology is the identification of mislabeled urine and blood samples based
upon the correct assessment of their blood groups.

We also analyze blood stain patterns for direction, distance, velocity, and

As the explanations do tend to become long and involved, please call us

regarding your needs in forensic serology.

Forensic Serology

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

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

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").

Body fluids and waste products

Blood cells and blood typing

Blood consists of a variety of different cell types, collectively known as blood

cells, which are suspended in a watery fluid called serum. Traditionally, the
study of blood is referred to as haematology whilst the study of serum (and
in par ticular the immune factors within it) is called serology. Typically, the
intrusion of forensic science has complicated things and the term ‘forensic
serology’ is sometimes taken to encompass not only the study of serum but
also blood cells, saliva and semen for forensic purposes. Mature human blood
cells can be divided into those that posses a nucleus and those that do not:
both types can provide forensic information. Those lacking a nucleus are the
red blood cells (RBC) (also known as erythrocytes) and the platelets. The red
blood cells possess the pigment molecule haemoglo- bin that gives blood its
red colour and is responsible for the transport of oxygen and carbon dioxide.
The platelets are smaller than the red blood cells and, unlike them, they are
capable of amoeboid-like movement, i.e. they move by sending out cell
processes in a similar manner to the single-celled organism ‘amoeba ’ and
consequently often look star-shaped when viewed using a microscope.
Platelets are responsible for the clotting mechanism and the production of
chemical growth factors that maintain the integrity of the blood vessels. The
cells that contain nuclei are collectively known as white blood cells (WBC) or
leucocytes. There are many different sorts of white blood cell but all of them
are capable of amoeboid-like movement and they are responsible for the
body’s immune defence capabilities. All the white blood cells contain nuclei
and they also have mitochondria – subcellular organelles that produce
energy for the cell therefore, unlike the red blood cells, they posses DNA. All
cells within the body carry upon their outer surface an array of molecules
called ‘antigens’. These antigens act like a passport and identify the cell as a
legitimate ‘citizen of the body’. The antigens are recognized by a group of pro
teins, called ‘antibodies’, that are secreted by certain white blood cells and
serve as ‘immigration control’. If a cell does not posses the correct antigens
on its surface, it is deemed to be a foreigner and an immune response is
mounted to destroy it. The nature of the antigens found on the surface of red
blood cells is of medical importance because unless the correct blood type is
used during a blood transfusion it would be rejected by the recipient ’s body
and thereby result in fatal consequences. The most common antigens found
on red blood cells are those that comprise the ABO system. The ABO system
works as follows: an individual’s red blood cells may posses either only class
A antigens (type A), only class B antigens (type B), both classes A and B
antigens (type AB) or neither class A nor class B antigens (type O). Persons
who are type A tolerate their own class A antigens but produce antibodies
against B antigens that bind to the surface of the red blood cells and form
bridges between them. This causes the red blood cells to agglutinate (clump
together) – a reaction that occurs quickly and can be observed using a
microscope. In a similar manner, type B persons produce antibodies against
class A antigens, type AB persons produce antibodies against neither class A
nor class B antigens and type O persons produce antibodies against both
class A and class B antigens. There are many other groups of antigens that
are found on red blood cells, the most well known being those responsible for
the Rhesus (Rh) factor (also known as antigen-D and agglutinogen-D). The
Rhesus factor is so called because the antigens responsible for it were first
described in Rhesus monkeys. Persons who posses these antigens are said to
be Rhesus positive [Rh(+)] whilst those who do not are Rhesus negative
[Rh(−)]. Eighty-three per cent of the UK population are Rh(+) and most
belong to either blood types O (44 per cent) or A (42 per cent). Although we
talk of a person being blood types A, B, AB or O and being Rh(+) or Rh(−), in
actual fact all of these characteristics can themselves be divided up into
many more sub-combinations (e.g. O1 and O2). Certain races tend to have
higher proportions of particular blood groups than others – for example type
AB is more common among the Japanese (10 per cent) than among
Europeans (UK, 4 per cent). More precise details of racial origin may be
obtained from finding evidence of rare inherited disease traits or particular
antigens. For example, sickle cell anaemia occurs almost exclusively among
Black people of African descent. Similarly, there is a variety of the Duffy
antigen (called phenotype a-b-) that is extremely common among West
Africans and their descendents but almost absent from White and Asian
populations. Both of these variations are considered to be protective against
malaria because the Plasmodium parasite finds it harder to recognize and
invade red blood cells expressing these traits. By contrast, the Kell antigen is
predominantly found
among White persons (Reid and Lomas-Francis, 1996). In addition to blood
type and the Rhesus factor, many of the enzymes and other proteins found
in red blood cells are also polymorphic, i.e. they exist in more than one form.
Consequently, even though an individual may have a common blood type
such as type O, once all the other variables are taken into account his
combination may be shared by only a small number of the population. If a
total of eight serological variables are used, it is generally estimated that the
chances of two unrelated persons sharing the same profile is between 0.01
and 0.001 (i.e. between 1 in 100 and 1 in a 1000). This estimate is known as
the match probability (Pm). This means that blood typing can provide a
means of iden tification, although it is not as accurate as DNA profiling in
which match probabilities of up to 10−13 have been claimed. Matching a
person’s blood profile to traces found at a crime scene might be
incriminating but on its own would be insufficient to prove guilt. It is,
however, effective at excluding suspects and therefore allowing the police to
concentrate their resources on more profitable lines of enquiry. Blood typing
suffers from several further drawbacks as a forensic tool. Firstly, by
comparison with DNA profiling, it requires relatively large amounts of sample
and is therefore of limited use where only small specks of blood are
available. Serological markers degrade quickly and conse quently the
amount of information that can be obtained from either a body or a
bloodstain that is several days old is reduced. The procedure is further
compromised by the interference caused by enzymes released by
contaminative bac-

teria. Interference can also be a problem where the bloodstain contains both
the assailant’s and victim’s blood (e.g. after a fight), if one of the parties has
recently received a blood transfusion (in which case their blood profile may
betemporarily altered) or following a rape in which semen is left inside the
victim’s body and therefore will be diluted by his or her serological profile. In
addition, the investigation may require a suspect to provide a blood sample.
This is an invasive medical procedure with which he or she may legitimately
refuse to cooperate.

Confirming the presence of blood

Bloodstains are not always obvious because of the manner in which they
were produced or because the assailant cleaned up the crime scene after
committing an assault. However, once blood is spilt, it can be extremely
difficult to remove all the traces. Any attempt at cleaning up inevitably
means using either the kitchen or bathroom as a ‘base’ – so it is a good place
to start looking for blood. In the cleaning process, blood can flow beneath
tiles or linoleum or between the boards of wooden flooring. Similarly, if a
water tap is grasped with a bloody hand, the blood may flow into the screw
mechanism. Consequently, blood can be detected in even an apparently
spotlessly clean room if one knows where to look. To highlight the presence
of blood an investigator sprays an indicator sub stance, such as luminol, onto
the suspect area. Luminol interacts with the iron found in haemoglobin to
produce a faint bluish glow that is best seen in dim light. Unfortunately,
luminol interacts with other iron-containing substances and also with copper
compounds. Consequently, whilst luminol is highly effective, and can
sometimes detect blood on clothing even after it has been washed, a positive
reaction only suggests the presence of blood and is not proof. Tests such as
this are therefore called ‘presumptive tests’. Presumptive tests are an
essential first step in a forensic investigation because many substances can
produce stains that give the appearance of blood. For example, there is said
to have been a case in which a child was reportedly raped in her own
bedroom but once the ‘bloodstains’ were analysed they turned out to be
caused by plum juice. The child apparently took a slice of plum tart from the
kitchen and ate it without permission in her bedroom. Presumably, clothing
or sheets became stained and the child then invented ‘an intruder’ to cover
up the ‘crime’. There are a number of spot tests that can determine whether
a suspicious stain is actually blood but the Kastle-Meyer test is the most
commonly used. In this test the stain is treated with a mixture of ethanol (to
improve the sensitiv ity of the test), phenolphthalein (a colour indicator) and
hydrogen peroxide (an oxidant). The hydrogen peroxide interacts with the
haem molecule of haemoglobin and is broken down into water plus highly
reactive free oxygen radicals these radicals then interact with the
phenolphthalein resulting in the solution changing from colourless to pink. In
order to be sure the test is working, one should always perform a negative
control (e.g. a spot of distilled water) and a

positive control (e.g. a spot of dried animal blood) immediately before assess
ing an unknown stain. If this is not done then the results could easily be
called into question. The Kastle-Meyer reaction is also subject to false
positives as a result of the hydrogen peroxide being broken down by
interactions with other chemicals these can be found in a variety of
substances of which, for some unknown reason, potatoes and horseradishes
are the most commonly quoted. Consequently, further tests would be
required to improve the reliability of the evidence. Even if blood is detected,
it is not immediately certain that it came from a human being. Household
pets are just as likely to fight, scratch and otherwise injure themselves as
any human and therefore leave their bloodstains on upholstery and clothing.
Similarly, when handling raw meat in the kitchen it is not unusual to leave
bloodstains upon surfaces and clothing. Furthermore, it is important to hunt
for animal blood when investigating crimes such as badger baiting or when a
pet was injured or killed during the course of a break-in or homicide. To
determine whether blood comes from a human or an animal one usually
requires a precipitin test, which involves reacting the blood sample with anti-
human antibodies – these are available commercially and are raised in
rabbits. The blood samples and the antisera (anti-human antibodies) are
placed in wells punched into an agar gel that is spread over a glass dish or
slide. The samples move towards one another through the agar by diffusion
or the process can be speeded up using an electric current. If a white line –
called the precipitin line – is formed at the point at which the two samples
meet, this is indicative of an interaction between the antigens in the blood
and antibodies in the rabbit antisera and therefore the blood is human. If no
precipitin line forms and there is a suspicion that the blood belongs to an
animal, then the procedure can be repeated using antibodies raised against
the appropriate animal sera. DNA-based techniques are now being developed
to differentiate human and non-human blood and tissue samples. For
example, Matsuda et al. (2004) describe a highly specific PCR-based protocol
(see later for more information on PCR) that utilizes primers for the human
mitochondrial cytochrome b gene. They state that following the agarose
electrophoresis step of the PCR process, human DNA produces a single band
whilst blood from other vertebrates fails to produce any bands at all.
Molecular methods such as this have the advantages of being simple,
extremely specific and require extremely small amounts of sample material
but the reagents tend to be expensive.


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

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.

As you can see from the graphic of this microscopic

image, 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

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% A+ 35% B+ 8% AB+ 4%
O- 6% A- 5% B- 2% 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
PGM 2-1 phosphoglucomutase
polymorphism in blood: Hb,
EAP erythrocyte acid phosphatase
which causes sickle-cell
EsD esterase D
anemia. The following are
AK adenyl kinase
some common
ADA adenoisine deaminase
GPT glutamic pyruvate transaminase
6-PGD 6-phosphogluconate dehydrogenase Each of these protein and
G-6-PD glucose-6-phosphate enzyme variants, as well as all
Tf dehydrogenase blood subtypes, have known
transferrin 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


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

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


Semen is a human body fl uid present in human males. It is a
viscid mucilaginous fl uid with faint yellow colour and
characteristic odour called seminal odour.

Volume is about 3 ml per ejaculate.

No. of Spermatozoa= 50,000 to 3,50,000 per ml. (10% of SP).
It is a suspension of spermatozoa in seminal plasma.

Figures 1-4 Showing Morphology of Human Sperm

Semen Composition

1. Spermatozoa (10%)
2. Seminal Plasma (90%)
3. Epithelial Cell (< 1%)

The Spermatozoa is produced in the testis by the process of

spermatogenesis. Spermatozoa contains lipid, proteins like
protamine & histone etc. and enzymes like dehydrogenises &
The total length of spermatozoa is about 50 Microns. it consist of
head and tail. The head is fl at, oval shaped - 4.6 X 2.6 X 1.6
Microns. in Length Width Thickness. The nucleus, which occupies
major portion of the head. The tail portion is responsible for the
movement of sperm.

The seminal plasma is a mixture of secretion derived from the

male accessory reproductive organs like epididymis, seminal
vesicles, the prostate, vasa-deferantia, bulbourethral & urethral
glands. The seminal plasma contains Citric Acid, Ascorbic Acid,
Lactic Acid, Fructose, potassium Choline Phosphate, Proteases,
free Amino Acids, Ergothioniene, Zinc, Calcium, Spermine, Lipids,
Enzymes like Fibrinogenase, Diastase, Acid & Alkaline
Phosphatase, Glysidases, a & ß Mannosidases a & ß Glucosidases,
ß Givcouridases.


Rape, Sodomy (Anal intercourse), Bestiality (Sexual intercourse by
a human being with a lower animal like dogs, calves, sheep
etc.),In case of false Accusation by a women , Incest (Sexual
intercourse in blood relation) and Sexual Murders.

Where to look for Seminal Stains

1. Clothes : Underwear, Bed sheet, Carpet, Towel, Pillow cover.
2. Body :Perineum, thigh, Vagina & pubic hair.
3. Seen of crime : On the fl oor or grass etc.

Method of Collection
Handling of articles bearing stains should be done very carefully
to avoid damage to spermatozoa.
Vaginal / anal / penile swabs should be sent along with their
smears on slides. Swabs should be taken on sterile gauze / cloth
and their smears prepared on sterile slides. These should be dried
in air at room temperature (37 degrees celcius) and swabs
dispatched in sterile test tube and slides in clean wrappers.

Methods Applied for Detection of Seminal Stains are Classifi ed as:

1. Physical Examination
2. Chemical Examination
3. Microscopic Examination

Physical Examination: Include Visual Examination. To naked eye

seminal stains generally appear translucent or opaque spots, at
times with yellowish tint and darker border depending on colour
and thickness of substrata, which, if absorbent, also acquire
stiff ness due to dried semen. On good substrata seminal stains
may appear to be fl uorescent under ultraviolet light.

Chemical examination:
PH = 7.4 Alkaline.
The tests used to detect Seminal Stains are:
1. Florence Test
2. Barberio Test
3. Acid Phosphatase Test
4. LDM Isoenzyme Method
5. Acid Phosphatase Isoenzyme Test
6. Creatinine in Phosphokinase
7. Ammonium Molybdate Test (Phosphorus)
8. Semen Specifi c Glycoprotein (P30 ) Test
9. Enzyme-linked immunosorbent assay (ELISA), the SEMA® assay,
for a seminal vesicle-specifi c antigen (SVSA)
Florence Test
Basis: Choline is detected in this method.
Procedure: A few drops of watery solution of the stain is extracted
and taken on a slide and a drop of Florence reagent (8%) W/V
solution of Iodine in water containing 5% W/V of Potassium Iodide)
is poured & allowed to mix slowly under a cover slip. Dark brown
crystals of choline periodide, generally needle shaped, formed
with a few minutes. Non-specifi cs & false negative results are

Berberio ’ s Test:
Basis: Detection of Spermine
Procedure: A few drops of Berberio ’ s reagent when added to
spermatic fl uid produces crystals of sperm in picrate (needle
shaped, rhombic & of yellow colour).
For various valid reasons, like non-specifi city and lack of
reproducibility, the fl orence and berberio ’ s tests have not been
accepted universally.
Modifi ed Fishman and Lerner ’ s method. The fl uid obtained after
thorough maceration of a small cloth piece (about 4mm2 ) is
placed in a cavity on a porcelain tile land two drops each of
citrate buff er (Ph 4.9) and 1% W/V aqueous solution of disodium
Phenyl phosphate are added. After 10 minutes the phenol is
detected by the addition of 2 drops of phenol reagent & 2 drops of
20% W/V solution of sodium carbonate. Blue colour developed
which indicates the presence of acid phosphatase.
Detection of Spermatozoa
Procedure: Seminal stains are extracted with 1 ml of water. 0.25
ml of clear extract is mixed with 0.25 ml of 40% W/V of sucrose.
0.1 ml of this mixture is subjected to vertical polyacryl amide gel
electrophoresis. Electrophoresis is carried out in refrigerators for
150 minutes using a current of 5 m A Isoenzyme bands are
revealed by staining.
This method gives a specifi c biochemical detection of
spermatozoa in semen in the presence of Vagtinal Fluid, Blood,
Nasal Secretion, Saliva & Urine.
Procedure: Seminal stains are extracted with water and is used in
polyacrylmile gel Electrphoretic method followed by staining with
methyl belliferyl. Phosphate reagent enable the seminal acid
phosphatase to be distinguished from that of other substance like
vaginal secretions.
The method is suffi ciently specifi c & applicable to semen derived
from normal, oligospermic azoospermic & vasectemized
1. LDH isoenzyme is stable in stains for over 4 week.
2. Isoenzyme pattern of human is diff erent from that of animal.
3. Positive results are obtain in large number of cases.
4. Can diff erentiate from vaginal secretions on pattern of bands.
Bases: Detection of creatinine phosphokinase. Normal seminal
fl uid content - 385 - 14000 U of CPK/W.
Diagnosis: >400V of CPK / ml.
Adv: Enzyme is stable & can be demonstrated in old status of six
Bases: Unique combination of choline & spermine is present only
in semen.
Liquid semen & dried seminal stains can be identifi ed by a thin
layer chromatographic Technique.
1 ML of semen present can be detected by this method.
The Micro Scopic detection of the Seminal stains is based in
morphology of spermatozoa.
Microscopic detection of spermatozoa. Cloth pieces from diff erent
stains are taken in 0.5 ml of 0.01 N HCL in small test tubes placed
in a beaker containing water. After sonication for 5 minutes the
extracts and the cloth pieces are transferred to separate micro
scope slides and cloth pieces delicately teased with a needle.
Threads are removed and the residual liquid is gently evaporated
to dryness. Residue obtained is stained haematoxylin and eosin.
Fluorescence MicroScopy:
Is also used for detection of spermatozoa. It is based on the
principle that Y Chromo some is fl uorescent to quinacrine. With
this method it is possible to detect both intact spermatozoa as
well as the disconnected heads.
With the above review it is quite clear that although there are so
many test available for the detection of semen & sperms required
to examine in cases of rape, sexual murder, unnatural, sexual
off ences. These tests have their won limitations advantages and
disadvantages. For various valid reasons like non-specifi city and
lack of reproducibility the Florence and berberio ’ s tests have not
been accepted universally apart from positive Florence test can
also be obtained from other body tissue containing choline.
Negative Florence may be obtained from seminal stain in case the
choline content is low or in cases where the stains are
decomposed. However false positive test are not obtained in cases
of vaginal secretion.
Acid phosphatase of prostatic origin is also utilized for the bio-
chemical detection of semen. Any interference of serum acid
phosphatase caused by the admixture of blood in the seminal
stains was till recently ruled out by taking - advantage of the fact,
that only prostatic acid phosphatase is inhibited by 1-tartrate.
This distinction, however, became invalid after the demonstration
by Willott in 1972 and Davies and Wilson in 1974, that vaginal
Acid phosphatase could also be inhibited by 1-tartrate. Apart from
these tests other tests like Radio-immuno assay and commercial
enzyme-linked immunosorbent assay (ELISA) are also available.
Commercial enzyme-linked immunosorbent assay (ELISA), the
SEMA® assay, for a seminal vesicle-specifi c antigen (SVSA)
provides highly sensitive detection of semen but these tests are
costly enough restricting their use for research purpose only.
The only methods among the available ones which appear to be
considered as specifi c and unambiguous, are the microscopic or
electrphoretic detection of spermatozoa in seminal stains derived
from normal individuals and the electrphoretic detection of
seminal Acid phosphatase in stains originating from all individuals
whether normal or abnormal.
ABO blood group system

ABO blood group antigens present on red blood cells and IgM
antibodies present in the serum

The ABO blood group system is the most important blood type
system (or blood group system) in human blood transfusion . The
associated anti-A antibodies and anti-B antibodies are usually IgM
antibodies, which are usually produced in the fi rst years of life by
sensitization to environmental substances such as food, bacteria
and viruses. ABO blood types are also present in some animals,
for example cows and sheep, and apes such as chimpanzees,
bonobos , and gorillas . [ 1 ]
 1 History of discoveries
 2 ABO antigens
 3 Serology
 4 Transfusion Reactions
 5 ABO hemolytic disease of the newborn
 6 Inheritance
 7 Distribution and evolutionary history
 8 Association with von Willebrand factor
 9 Subgroups
o 9.1 A1 and A2
 10 Bombay phenotype
 11 Nomenclature in Europe and former USSR
 12 Examples of ABO and Rhesus D slide testing method
 13 Universal blood created from other types, and artifi cial
 14 Myths
 15 References
 16 Further reading
 17 External links

History of discoveries
The ABO blood group system is widely credited to have been
discovered by the Austrian scientist Karl Landsteiner, who found
three diff erent blood types in 1900; [ 2 ] he was awarded the Nobel
Prize in Physiology or Medicine in 1930 for his work. Due to
inadequate communication at the time it was subsequently found
that Czech serologist Jan Janský had independently pioneered the
classifi cation of human blood into four groups, [ 3 ] but Landsteiner's
independent discovery had been accepted by the scientifi c world
while Janský remained in relative obscurity. Janský's classifi cation
is however still used in Russia and states of former USSR (see
below). In America, Moss published his own (very similar) work in
1910. [ 4 ]
Landsteiner described A, B, and O; Decastrello and Sturli
discovered the fourth type, AB, in 1902. [ 5 ] Ludwik Hirszfeld and E.
von Dungern discovered the heritability of ABO blood groups in
1910 – 11, with Felix Bernstein demonstrating the correct blood
group inheritance pattern of multiple alleles at one locus in 1924.
. Watkins and Morgan, in England, discovered that the ABO
epitopes were conferred by sugars, specifi cally N-
acetylgalactosamine for the A-type and galactose for the B-type
(Morgan, W. T. J. & Watkins, W. M. Br. Med. Bull. 25, 30 – 34 (1969),
Watkins, W. M. in: Advances in Human Genetics Vol. 10 (eds
Harris, H. & Hirschhorn, K.) 1 – 136 (Plenum, New York, 1980),
Watkins, W. M. & Morgan, W. T. J. Vox Sang. 4, 97−119 (1959).
After much published literature claiming that the ABH substances
were all attached to glycosphingolipids, Laine's group (1988)
found that the band 3 protein expressed a long polylactosamine
chain (Jarnefelt, Rush, Li, Laine, J. Biol. Chem. 253:
8006 – 8009(1978)) which contained the major portion of the ABH
substances attached (Laine and Rush in Molecular Immunology of
Complex Carbohydrates (A. Wu, E. Kabat, Eds.) Plenum Publishing
Corporation, N.Y. NY (1988)). Later, Yamamoto's group (Yamamoto,
et al., Nature 345, 229 – 233 (1990)), showed the precise glycosyl
transferase set that confers the A, B and O epitopes.
[edit] ABO antigens

Diagram showing the carbohydrate chains which determine the

ABO blood group
The H antigen is an essential precursor to the ABO blood group
antigens. The H locus is located on chromosome 19. It contains 3
exons that span more than 5 kb of genomic DNA, and it encodes a
fucosyltransferase that produces the H antigen on RBCs. The H
antigen is a carbohydrate sequence with carbohydrates linked
mainly to protein (with a minor fraction attached to ceramide
moiety). It consists of a chain of β-D- galactose, β-D-N-
acetylglucosamine , β-D-galactose, and 2-linked, α-L- fucose, the
chain being attached to the protein or ceramide.
The ABO locus is located on chromosome 9. It contains 7 exons
that span more than 18 kb of genomic DNA. Exon 7 is the largest
and contains most of the coding sequence. The ABO locus has
three main alleleic forms: A, B, and O. The A allele encodes a
glycosyltransferase that bonds α- N-acetylgalactosamine to D-
galactose end of H antigen, producing the A antigen. The B allele
encodes a glycosyltransferase that joins α-D-galactose bonded to
D-galactose end of H antigen, creating the B antigen.
In case of O allele the exon 6 contains a deletion that results in a
loss of enzymatic activity. The O allele diff ers slightly from the A
allele by deletion of a single nucleotide - Guanine at position
261. The deletion causes a frameshift and results in translation of
an almost entirely diff erent protein that lacks enzymatic activity.
This results in H antigen remaining unchanged in case of O
The majority of the ABO antigens are expressed on the ends of
long polylactosamine chains attached mainly to Band 3 protein ,
the anion exchange protein of the RBC membrane, and a minority
of the epitopes are expressed on neutral glycosphingolipids .
[edit] Serology
Anti-A and anti-B antibodies (called Isohaemagglutinins ), which
are not present in the newborn, appear in the fi rst years of life. It
is possible that food and environmental antigens (bacterial, viral
or plant antigens) have epitopes similar enough to A and B
glycoprotein antigens. The antibodies created against these
environmental antigens in the fi rst years of life can cross react
with ABO -incompatible red blood cells when it comes in contact
with during blood transfusion later in life. Anti-A and anti-B
antibodies are usually IgM type, which are not able to pass
through the placenta to the fetal blood circulation. O -type
individuals can produce IgG-type ABO antibodies.
The "Light in the Dark theory" (DelNagro, 1998) suggests that
when budding viruses take with them host cell membranes from
one human patient (in particular from the lung and mucosal
epithelium where they are highly expressed) they also take along
ABO blood antigens from those membranes, and may carry them
into secondary recipients where these antigens can elicit a host
immune response against these non-self foreign blood antigens.
These viral-carried human blood antigens may be responsible for
priming newborns into producing neutralizing antibodies against
foreign blood antigens. Support for this theory has come to light
in recent experiments with HIV. HIV can be neutralized in "in-vitro"
experiments using antibodies against blood group antigens
specifi cally expressed on the HIV producing cell lines. [7] [8]

The "Light in the Dark theory" suggests a new novel evolutionary

hypothesis that there is true communal immunity, which has
developed to reduce the inter-transmissibility of viruses within a
population. It suggests that individuals in a population supply and
make a diversity of unique antigenic moieties so as to keep the
population as a whole more resistant to infection. A system set up
ideally to work with variable recessive alleles.
Transfusion Reactions
Due to the presence of isoantibodies against non self blood group
antigens, individuals of type A blood group immediately raises
anti-B antibodies against B-blood group RBCs if transfused with
blood from B group. The anti-B antibodies bind to B antigens on
RBC and causes complement -mediated lysis of the RBCs. The
same happens for B and O groups (which raises both anti-A and
anti-B antibodies). However only blood group AB does not have
anti-A and anti-B isoantibodies. This is because both A and B-
antigens are present on the RBCs and are both self antigens,
hence they can receive blood from all groups and are universal
 Individuals with type A blood can receive blood from donors
of type A and type O blood.
 Individuals with type B blood can receive blood from donors
of type B and type O blood.
 Individuals with type AB blood can receive blood from donors
of type A, type B, type AB, or type O blood.
 Individuals with of O blood can receive blood from donors of
only type O.
 Individuals of type A, B, AB and O blood can receive blood
from donors of type O blood. Type O - blood is called the
universal donor .
One caveat to this axiom of 'universal donor' is that this applies
to packed RBC's and not to whole blood products. Using the fi rst
table, type O carries anti-A and anti-B antibodies in the serum. To
transfuse a type A, B, or AB recipient with type O whole blood
would produce a hemolytic transfusion reaction due to the
antibodies found in the serum of whole blood.
A A or O
B B or O
A, B, AB, or
No antibodies are formed against the H antigen, except in those
individuals with the Bombay phenotype.
In ABH secretors, ABH antigens are secreted by most mucus-
producing cells of the body interfacing with the environment,
including lung, skin, liver, pancreas, stomach, intestines, ovaries
and prostate. [ 9 ]

ABO hemolytic disease of the newborn

Main article: Hemolytic disease of the newborn (ABO)
ABO blood group incompatibilities between the mother and child
does not usually cause hemolytic disease of the newborn (HDN)
because antibodies to the ABO blood groups are usually of the IgM
type, which do not cross the placenta; however, in an O -type
mother, IgG ABO antibodies are produced and the baby can
develop ABO hemolytic disease of the newborn .
[edit] Inheritance

A and B are codominant, giving the AB phenotype.

Blood group inheritance
O O O, A O, B A, B
O, O, A, B, A, B,
A O, A
O, O, A, B, A, B,
B O, B
A, A, B,
AB A, B, AB A, B, AB
Blood groups are inherited from both parents. The ABO blood type
is controlled by a single gene with three alleles: i, I A , and I B . The
gene encodes a glycosyltransferase — that is, an enzyme that
modifi es the carbohydrate content of the red blood cell antigens.
The gene is located on the long arm of the ninth chromosome
The I A allele gives type A, I B gives type B, and i gives type O. As
both I A and I B are dominant over i, only ii people have type O
blood. Individuals with I A I A or I A i have type A blood, and individuals
with I B I B or I B i have type B. I A I B people have both phenotypes,
because A and B express a special dominance relationship:
codominance , which means that type A and B parents can have an
AB child. A type A and a type B couple can also have a type O
child if they are both heterozygous ( I B i,I A i) Therefore, an O child is
not a direct proof of illegitimacy, just as a child with blond hair
could be born from parents who both had brown hair. The cis-AB
phenotype has a single enzyme that creates both A and B
antigens. The resulting red blood cells do not usually express A or
B antigen at the same level that would be expected on common
group A 1 or B red blood cells, which can help solve the problem of
an apparently genetically impossible blood group. [ 1 0 ]
Distribution and evolutionary history
The distribution of the blood groups A, B, O and AB varies across
the world according to the population. There are also variations in
blood type distribution within human subpopulations.
In the UK, the distribution of blood type frequencies through the
population still shows some correlation to the distribution of
placenames and to the successive invasions and migrations
including Vikings , Danes, Saxons, Celts, and Normans who
contributed the morphemes to the placenames and the genes to
the population. [ 1 1 ]
There are six common alleles of the ABO gene that produce one's
blood type: [ 1 2 ] [ 1 3 ]
 A101 (A1)  B101 (B1)  O01 (O1)
 A201 (A1)  O02 (O1v)
 O03 (O2)
Many rare variants of these alleles have been found in human
populations around the world.
Some evolutionary biologists theorize that the I A allele evolved
earliest, followed by O (by the deletion of a single nucleotide,
shifting the reading frame) and then I B . [ c i t a t i o n needed]
This chronology
accounts for the percentage of people worldwide with each blood
type. It is consistent with the accepted patterns of early
population movements and varying prevalent blood types in
diff erent parts of the world: for instance, B is very common in
populations of Asian descent, but rare in ones of Western
European descent.) Another theory states that there are four main
lineages of the ABO gene and that mutations creating type O have
occurred at least three times in humans [ 1 4 ] . From oldest to
youngest, these lineages comprise the following alleles:
A101/A201/O09, B101, O02 and O01. The continued presence of
the O alleles is hypothesized to be the result of balancing
selection [ 1 4 ] . Both theories, contradict the previously-held theory
that type O blood evolved earliest, supported by the fact that all
human beings can receive it [ c i t a t i o n needed]
. The British National Blood
Transfusion Service states this to be the case (see the web-link
under External Links below) and says that originally all human
beings were type O.
Association with von Willebrand factor
The ABO antigen is also expressed on the von Willebrand factor
(vWF) glycoprotein , [ 1 5 ] which participates in hemostasis (control of
bleeding). In fact, having type O blood predisposes to bleeding, [ 1 6 ]
as 30% of the total genetic variation observed in plasma vWF is
explained by the eff ect of the ABO blood group, [ 1 7 ] and individuals
with group O blood normally have signifi cantly lower plasma levels
of vWF (and Factor VIII) than do non- O individuals. [ 1 8 ] [ 1 9 ] In addition,
vWF is degraded more rapidly due to the higher prevalence of
blood group O with the Cys1584 variant of vWF (an amino acid
polymorphism in VWF): [ 2 0 ] the gene for ADAMTS13 (vWF-cleaving
protease) maps to the ninth chromosome (9q34), the same locus
as ABO blood type. Higher levels of vWF are more common
amongst people who have had ischaemic stroke (from blood
clotting) for the fi rst time. [ 2 1 ] The results of this study found that
the occurrence was not aff ected by ADAMTS13 polymorphism, and
the only signifi cant genetic factor was the person's blood group.
This section is incomplete - please help to expand it.
A1 and A2
The A Blood Type contains about twenty subgroups, of which A1
and A2 are the most common (over 99%). A1 makes up about 80%
of all A-type blood, with A2 making up the rest. [ 2 2 ] These two
subgroups are interchangeable as far as transfusion is concerned,
however complications can sometimes arise in rare cases when
typing the blood. [ 2 2 ]

Bombay phenotype
Individuals with the rare Bombay phenotype ( hh) do not express
antigen H on their red blood cells. As H antigen serves as
precursor for producing A and B antigens, the absence of H
antigen means the individuals do not have A or B antigens as well
(similar to O blood group). However, unlike O group H antigen is
absent, hence the individuals produce isoantibodies to antigen H
as well as to both A and B antigens. In case they recieve blood
from O blood group, the anti-H antibodies will bind to H antigen on
RBC of donor blood and destroy the RBCs by complement -
mediated lysis. Therefore Bombay phenotype can receive blood
only from other hh donors (although they can donate as though
they were type O).
Nomenclature in Europe and former USSR
In parts of Europe the "O" in ABO blood type is substituted with
"0" (zero), signifying the lack of A or B antigen. In the former
USSR blood types are referenced using numbers and Roman
numerals instead of letters. This is Janský's original classifi cation
of blood types. It designates the blood types of humans as I, II, III,
and IV, which are elsewhere designated, respectively, as O, A, B,
and AB. [ 2 3 ] The designation A and B with reference to blood groups
was proposed by Ludwik Hirszfeld .
Examples of ABO and Rhesus D slide testing method

Blood group O positive: neitherResult: Blood group B negative:

anti-A nor anti-B haveanti-A and anti-Rh have not
agglutinated, but anti-Rh has agglutinated but anti-B has
In the slide testing method shown above, three drops of blood are
placed on a glass slide with liquid reagents. Agglutination
indicates the presence of blood group antigens in the blood.
Universal blood created from other types, and artifi cial
In April 2007 an international team of researchers announced in
the journal Nature Biotechnology an inexpensive and effi cient way
to convert types A, B and AB blood into type O. [24]
This is done by
using glycosidase enzymes from specifi c bacteria to strip the
blood group antigens from red blood cells . The removal of A and B
antigens still does not address the problem of the Rhesus blood
group antigen on the blood cells of Rhesus positive individuals,
and so blood from Rhesus negative donors must be used. Patient
trials will be conducted before the method can be relied on in live
Another approach to the blood antigen problem is the creation of
artifi cial blood which could act as a substitute in emergencies.
There are numerous popular myths surrounding ABO blood groups.
These beliefs have existed since the ABO blood groups were
identifi ed and can be found in diff erent cultures throughout the
world. For example, during the 1930s, connecting blood groups to
personality types became popular in Japan and other areas of the
world. [ 2 5 ]
The popularity of Peter J. D'Adamo 's book, Eat Right For Your
Blood Type suggests that these myths persist. This book claims
that ABO blood groups can be used to determine diet as well as to
predict diseases.
Additional myths include the idea that Group A causes severe
hangovers, group O is associated with perfect teeth, and those
with blood group A2 have the highest IQs. Scientifi c evidence in
support of these concepts is scant or nonexistent.

Once a biological evidence has been identified, it is necessary to determine

whether or not it is of human origin; and if of non- human origin, then to what species it
belongs. The species specific proteins in the bloodstains or other body tissues may be
identified with the help of species specific antibodies.

The species specific proteins from the bloodstain or tissue are extracted in
normal saline (8.5 g of sodium chloride in one litre of distilled water) or 5% ammonia
solution. The latter is a better extractant for aged stains or stains on synthetic fibers and
calcified tissues.

3.1. Preparation of Extract

3.1.1. Preparation of Extract from Bloodstains

1. Take small portion of the bloodstain and dip in minimum amount of normal saline or
5% ammonia solution (especially for the older/fixed stains).

2. Keep for 1-2 hours to get light brown coloured solution.

3. Centrifuge and use the supernatant.

3.1.2. Preparation of Extract from Calcified/keratinized Tissues

1. Pulverize the bone/dental/nail tissue thoroughly to obtain a very fine powder.

2. Take about 100mg of the pulverized tissue and dip in minimum amount of 5%
ammonia solution.

3. Keep for overnight.

4. Centrifuge and use the supernatant.

3.1.3. Preparation of Extract from Soft Tissues

1. Take small portion of the tissue and add one drop of normal saline or 5% ammonia

2. Homogenise tissue thoroughly and keep for 1-2 hours.

3. Centrifuge and use the supernatant.

3.2 Precipitin Tube Method

1. Take six (can vary on the number of antisera used) precipitin tubes and place them
vertically in a precipitin tube stand and label.

2. Put a drop of the bloodstain/tissue extract in the tubes.

3. Carefully add one drop of antiserum for species origin (anti Human serum, anti Fowl
serum, anti Dog serum, anti Cow Serum, anti Goat serum etc.) along the walls of tube
and leave undisturbed for 30 minutes at room temperature.

4. Examine for a white ring at the interface of two solutions.

3.3. Double Diffusion

Both of the reactants, antigen and antibody diffuse towards each other in gel; and
when an antigen combines with its specific antibody at optimum proportions, a precipitin
arc forms.

1. Weigh 100 mg of Agar (or Agarose) and put in a beaker containing 10 ml of normal
2. Heat the solution while shaking

3. When the Agar gets dissolved completely, pour it over in a properly levelled
disposable petri dish or glass slide to make a 1-2mm thick agar layer. Wait until the agar

4. Punch wells in gel, each about 5 mm apart, in a hexagonal way.

5. Seal the bottoms of punched wells with dilute agar (0.5%)

6. Fill the central well with tissue extract and peripheral wells with different antisera for
species origin or vice versa (anti Human serum, anti Fowl serum, anti Dog serum, anti
Cow Serum, anti Goat serum etc.).

7. Cover the petri dish and keep gel in a moist chamber for overnight.

8. Examine gel for the presence of precipitin arcs.

3.4. Cross Over Electrophoresis

` In a buffered gel, the stain extract (antigen) is placed in the

cathodic well and the antiserum in the anodic one. The -globulin antibodies migrate
cathodically because of electroendosmosis, while the other serum proteins migrate
anodically when electric current is applied. A precipitin reaction takes place midway
between the paired wells when an antigen combines with its specific antibody.

1. Weigh 100 mg of Agar (or Agarose) and put in a beaker containing 10 ml of Gel

2. Heat the solution while shaking

3. When the Agar fully gets dissolved, pour it over in a properly levelled, clean glass
slide to make a 1-2mm thick agar layer. Wait until the agar solidifies.

4. Punch wells in pairs, each about 5 mm apart.

5. Using a pipette, fill the right-hand sample well of the pair with stain extract.

1. Fill the cathodic side well of the pair with stain extract and anodic well with species
specific antiserum (anti Human serum, anti Fowl serum, anti Dog serum, anti Cow
Serum, anti Goat serum etc.). See diagram for example.

Extract O O Anti Human Serum

Cathode Extract O O Anti Fowl Serum Anode
Extract O O Anti Dog Serum

2. Place the slide on the electrophoresis chamber. The stain extract should be nearest
to the cathode, the antiserum nearest to the anode.

3. Connect the gel to tank buffer chambers by two pieces of filter papers on each side.

4. Electrophorese for 20 minutes at 150 volts. Record conditions.

5. Following electrophoresis, switch off the power supply and remove the slide.

6. Observe the slide with the aid of a lamp. A fine white line of precipitate between a
pair of wells is a positive reaction.

Staining Procedure

1. Place the slide in saline overnight at room temperature. This is to wash

away any unreacted proteins.
2. Wash for 1 hour in distilled water at room temperature to remove any
3. Remove from water cover with a piece of damp filter paper and place in
the oven to dry.
4. Remove the filter paper when dry, and wash plate under running tap
water, gently rubbing gel surface to remove fragments of filter paper.
5. Place in Amido Black stain for 10 minutes.
6. Transfer to destain solution, examining periodically.
7. Remove when background is clear and precipitin bands are stained a
deep blue/black.
8. Allow to dry and double check your results.
Note: It should be noted that the results of cross-over electrophoresis are only as
accurate as the monospecificity of the antiserum used. Any precipitin bands
formed by cross-reaction could be interpreted as a false positive reaction.


TANK BUFFER* Sodium barbiturate 8.1g
(pH- 8.6) Diethylbarbituric acid 1.38g
Calcium lactate 0.39g
Add Distilled water to make
one Litre
GEL BUFFER* Sodium barbiturate 7.01g
(pH 8.6) Diethylbarbituric acid 1.38g
Calcium lactate 1.03g
Add Distilled water to make
one Litre
SUPPORT MEDIUM 1% Agar or agarose in Gel Buffer
TEMP. & CONDITIONS Room temperature
VOLTAGE & DURATION 150V for 20 minutes
STAIN (AMIDO Naphthalene black 10B 0.1 g
BLACK)# Methanol 50mL
Glacial acetic acid 10 mL
Distilled water 50mL
Glacial acetic acid
200 mL
Distilled water
1 Litre

*Barbital buffer can be substituted with the following Tris- glycine buffer:

Tank Buffer (pH- 8.4): 0.037 M Tris 4.50g

0.29M Glycine 21.8g
Dissolve in distilled water, adjust pH with HCl and make final
volume 1 litre.
Gel Buffer (pH- 8.4): Same as
tank buffer.
# Coomassie Brilliant Blue (R- 250) (1g in 500mL of Destain solution) stain can be used
instead of Amido Black.

4.1. Collection of Fresh Blood Sample:

1. Sterlize the bulb of a finger with an alcohol swab and let it dry.

2. Prick it with a disposable needle while compressing the finger bulb.

3. Collect the blood in a tube with normal saline.

4. Centrifuge the contents at about 3000rpm for one minute.

5. Discard the supernatant and resuspend the button of cells in fresh normal saline.

6. Repeat steps 4 and 5 twice and finally prepare 2% or 0.2% (as per requirement) cell
suspension in normal saline.

Note: Packed red cell suspension (50%) can be stored for upto a week in refrigerator.
The cells should be washed in normal saline prior to use.

4.2. Grading of the Agglutination Reaction Intensities

Macroscopic agglutination:

C - Complete, all cells forming one big clump.

V - Visual, all cells forming few visible big clumps.

Microscopic agglutination:
+++ - Very large agglutination clumps with few free cells.

++ - Smaller agglutinates with more free cells.

+ - Agglutinates of 5- 10 cells with many free cells.

W - Agglutinates of 3- 5 cells with many free cells.

N - No agglutination.

4.3. Preparation of Anti- H Lectin

1. Soak about 2g seed of Ulex europaeus in 10ML of normal saline for overnight.

2. Macerate seeds and agitate paste for an hour. Centrifuge at 3,000rpm for 5 minutes
and discard sediment.

3. If supernantant is cloudy, centrifuge at about 10,000rpm for 15minutes and use the

4. Check the specificity of the lectin with group O cells & titrate. Anti- H lectin with
minimum titre of 32 should be used for grouping reactions.

4.4. Grouping of Fresh Blood

4.4.1. ABO Grouping

ABO blood group can be determined from the fresh blood samples by detection of
antigens on RBC's (forward method) or detection of antibodies in serum (reverse
method). Forward Method

1. Take a clean, dry cavity tile and mark two cavities as A and B.

2. Put one drop of anti-A serum and anti-B serum in the above marked cavities

3. Add one drop of about 2% cell suspension in each cavity and mix the contents

4. Rotate the tile for 5 minutes and examine the contents macroscopically as well as
microscopically for agglutination.

Agglutination in cavity Antigen on RBC's Blood group


+ - A A

- + B B

+ + A, B AB

- - Nil O Reverse Method

1. Let the whole blood sample clot and then remove the serum carefully.

2. Take one clean and dry cavity tile and mark two cavities as A and B.
3. Put one drop of serum in each cavity.

4. Add one drop of 2% cell suspension of A and B cells in the respective cavities. Mix
the contents and rotate the tile for five minutes.

5. Examine the contents for the presence of agglutination macroscopically as well as


Agglutination in cavity Antibody present

Blood group

A B - + Anti B

+ - Anti A

- - Nil

+ + Anti A, Anti B O

In case of Bombay blood group, RBC's are not agglutinated by anti- A serum, anti- B serum or anti- H
lectin. Presence of anti- H in the serum is indicative of Bombay phenotype O h.

4.4.2 Rh Typing
1. Take one clean and dry cavity tile and mark five cavities as C, c, D, E & e.

2. Place one drop of anti C serum, anti c serum, anti D serum, anti E serum and
anti e serum in the respective cavities.

3. Add one drop of 2% cell suspension in each cavity and mix the contents
thoroughly. Rotate the tile for 5 minutes at room temperature.

4. Examine for agglutination both macroscopically as well as microscopically.

5. If there is no agglutination, keep the tile at 37 0 C for 15 minutes, rotate the

tile and again examine for agglutination.
Presence of agglutination indicates the presence of respective antigen.

4.4.3. MN Typing

1. Take a clean, dry cavity tile and mark two cavities as M and N.

2. Wash red blood cells three times with normal saline. Properly washed cells are
absolutely critical in MN typing in order to avoid false positive results.

3. Place one drop of the appropriate antiserum in the wells.

4. Add one drop of 2.0% suspension of red blood cells.

5. Rotate the tile at room temperature for five minutes.

6. Examine for agglutination both macroscopically as well as microscopically.

Agglutination in cavity Antigen on RBC's Blood group


+ - M M

- + N N

+ + M, N MN

4.4.4. Lewis Typing

1. Label two test tubes: Lea and Leb.

2. Place one drop of anti Lea serum and anti Leb serum in the respective tubes.
3. Add one drop of 2.0% cell suspension in the both tubes and mix.

4. Allow tubes to incubate at room temperature for 30 minutes.

5. Centrifuge at about 5000rpm for 15 seconds.

6. Gently shake the tube until the cell button just detaches itself from the bottom of the
tube in one or more agglutinated clumps or until the cell button disperses.

7. Read and grade the results macroscopically as well as microscopically.

Agglutination in cavity Antigen on RBC's Blood group

Lea Leb

+ - Lea Le a+b-

- + Leb Le a-b+

- - Nil Le a-b-

4.5. ABO Grouping of Body Fluid Stains and Tissues

If microbial growth is present over the stains, the stains can be kept at 100 0C for
one hour to destroy the microbes and then typing can be performed.

The polyclonal antisera are preferred over monoclonal antisera for typing the
body fluid stains & tissues. If polyclonal antisera are not available, monoclonal antisera
from different sources should be pooled and validated before analyses.

4.5.1. Reverse Grouping of Bloodstains: Lattes test

This method is based on the identification of antibody in the blood stains. It is
useful in the identification of mixture of blood stains.

1. Take about 0.5 sq.cm bloodstain and add 2 drops of normal saline. Keep for an hour.

2. Squeeze the stain and then remove it. Centrifuge the extract and take the

3. Take one clean dry cavity tile and mark two cavities as A and B.

4. Put one drop of bloodstain extract in each cavity.

5. Add one drop of 2% cell suspension of A and B cells in the respective cavities. Mix
the contents and rotate the tile for five minutes.

6. Examine the contents for the presence of agglutination macroscopically as well as


Agglutination in cavity Antibody present Blood group


- + Anti B A

+ - Anti A B

- - Nil AB

+ + Anti A, Anti B O

Precaution: The antibodies are less stable in comparison to antigens and can be typed
only in the fresh stains. The method is less sensitive also.

4.5.2. Absorption Elution Method

1. Take three clean and dry test tubes and mark them A, B and H.
2. Cut the blood stain and put about 2 sq. mm or 2- 5mm long threads in each test tube.

3. Dip the fabric in anti- A serum, anti- B serum and anti- H lectin respectively and keep
at 40C for overnight.

4. Then remove the antiserum and give 3-4 washings with ice chilled normal saline.

5. After the last wash remove whole of the normal saline and add one drop of fresh
normal saline.

6. Plug the test tubes with cotton swab and keep in water bath at 56-60 0C for 15- 20

7. Add one drop of 0.2-0.5% A, B and O indicator cells in the respective tubes and keep
at 40C for half an hour.

8. Centrifuge, shake and examine the contents for agglutination both macroscopically
and microscopically.

Agglutination in cavity Blood group


+ - - or + A

- + - or + B

+ + - or + AB

- - + O

Precaution: 1. Washing should be done with ice chilled normal saline.

2. At the time of elution, only one drop of normal saline is required.

4.5.3. Absorption Elution Method: Howard Martin

1. Take a cellulose acetate sheet (minimum thickness 0.4mm) and mark three areas as
A, B and H. Glue one cm long bloodstained thread with acetone/fingernail polish to each
of 3 areas.

2. Allow the threads to fix on the sheet for 15 minutes.

3. Put one drop of the anti- A serum, anti- B serum and anti- H lectin respectively on the
fixed threads

4. Place the sheet in a moist chamber and allow it to absorb overnight at 4 0C in the

5. Remove from the refrigerator, rinse off the excess antisera and blot the threads dry
with a paper towel by inverting the plate face down on a paper towel and rubbing the
back of the glass plate with another towel.

6. Place in the refrigerator at 40C for a minimum of 2 hours. Longer wash times will not
have a negative effect on the results provided the temperature does not exceed 4 0C.
Shorter wash times may result in incomplete rinsing of unbound antibody.

7. Remove from the wash bath and blot dry with a paper towel as previously described.

8. Add one drop of appropriate 0.2- 0.5% indicator cells to each thread.

9. Place in a moist chamber and elute in a 56 0C incubator for 20 minutes.

10. Place the sheet in a moist chamber and rotate on a VDRL rotator for 30 minutes.

11. Read results microscopically.

4.5.4. Absorption Elution: Ammonia Method

Ammoniacal elution is especially useful when typing bloodstains on substrates

which do not lend themselves to Howard-Martin absorption elution typing or very old,
insoluble bloodstains.

1. Extract a 5 x 5 mm stain with 4-5 drops of 5% ammonia solution. Extract should be

straw coloured. (In this method, a lighter extract works considerably better than a darker

2. Place one drop of the extract in each of three wells of a serological slide marked A, B,
and H.

3. Heat-fix the extract for a minimum of one hour at 56 0C.

4. Add one drop of anti- A serum, anti- B serum and anti- H lectin respectively to each
well and allow to absorb for five minutes in a moisture box at room temperature.

5. Quickly rinse off the antiserum and place in a normal saline container kept in
refrigerator at 40C for 10 minutes. Saline should be changed frequently.

6. Carefully blot dry each well.

7. Add one drop of appropriate 0.2- 0.5% cell suspension (in 1.5% normal saline
albumin) to each well.
8. Place slides in a moist chamber at 370C for 15 minutes for the elution process.

9. Transfer the slide to a room temperature moist chamber and rotate for ten minutes.

10. Read results microscopically, grade, and record.

4.5.5. Absorption Elution Method Using Papainised Cells

Minute amount of bloodstains, calcified tissues (bone & teeth) and keratinized
tissues (hair & nail) can be typed by treating indicator cells with papain. This increases
the sensitivity of the method manifold. The method is similar to as given for the grouping
of bloodstains by Absorption- Elution method, except papainised cell suspension is used
instead of the untreated ones. Preparation of Papain Solution

1. Add 200 mg of papain powder to 10 mL of normal saline and mix thoroughly.

2. Filter the solution and centrifuge filtrate at 10,000rpm for 10 minutes.

3. Store the supernatant at 40C. Papain Treatment of the Cells

1. Centrifuge the indicator cells to get a button of cells. Remove the supernatant and
add papain solution twice in volume.

2. Keep at 370C for 10 minutes.

3. Then give three washings to the cells with normal saline alongwith centrifugation and
prepare the required percentage

Always use freshly prepared papainised cells for grouping.

4.5.6 Grouping of Keratinized Tissues (Hair Shaft or Nail)

1. Clean the sample by dipping in mixture of ethanol: ether (1:1).

2. Dry and flatten them upto 4-5 times by hammering. The keratinized tissue should be
flattened thoroughly to increase the surface area.

3. Take about 1 cm of the flattened strand or 10 mg of nail in each tube marked A, B and
H and follow the Absorption elution method employing papainised indicator cells.

4.5.7. Grouping of Calcified Tissues (Bone or Dentine)

1. Pulverize the tissue to obtain a very fine powder.

2. Put 5-10 mg of the tissue in each tube and follow absorption elution method
employing papainised indicator cells.

4.5.8. Mixed Agglutination Method

1. Take three clean and dry test tubes and mark them A, B and H.

2. Cut the blood stain and put about 2 sq. mm or 2- 5mm long threads in each test tube.

3. Dip the fabric in anti- A serum, anti- B serum and anti- H lectin respectively and keep
at 40C for overnight.
4. Then remove the antiserum and give 3-4 washings with ice chilled normal saline.

5. After the last wash remove whole of the normal saline and add one drop of 0.2-0.5%
A, B and O indicator cells in the respective tubes.

6. Plug the test tubes with cotton swab and keep in water bath at 50 0C for 10 minutes.

7. Keep tubes at 40C for half an hour, centrifuge, shake and examine the contents for
agglutination attached to fabrics, both macroscopically and microscopically.

Agglutination in cavity Blood group


+ - - or + A

- + -or + B

+ + -or + AB

- - + O

Precaution: Washing should be done with ice chilled normal saline.

4.6. ABO Groping of Saliva and Semen

The persons who secrete ABH substances in their saliva, semen or vaginal
secretions and urine etc. are called secretors and ABO blood group can be detected
from their body fluids and stains. All secretors secrete H substance in the body fluids
irrespective of their blood group.

ABO grouping from these body secretions should be done by Absorption-

Inhibition as well as Absorption- Elution method as detailed earlier.
4.6.1. Typing of Fresh Saliva

Fresh saliva has enzymes which degrades the ABH substances. Hence, keep the tube
containing fresh saliva in boiling waterbath for about 10 minutes to destroy the enzyme.
Before typing, dilute the fresh saliva 2-3 times with normal saline.

4.6.2. Preparation of Extract from semen or saliva stains

1. Cut about 0.5- 1.0sq.cm of the stain and dip in 4-5 drops of the normal saline.

2. Keep at 40C for a minimum of two hours.

3. Squeeze out the stain fabric and centrifuge the contents at about 5000rpm for 2
minutes. Use the supernatant for grouping test.

4.6.3. Determination of Titre

The determination of the titres of anti-A serum, anti-B serum and anti-H lectin is
the prerequisite for determining the secretor status.

1. Take a clean and dry cavity tile and mark the cavities 1 to 12.

2. Place one drop of the antiserum in cavities marked 1 and 2.

3. Place one drop of the normal saline in cavities marked 2 to 12.

4. Mix the contents in 2nd cavity and transfer one drop to 3rd cavity.

5. Mix the contents in 3rd cavity and transfer one drop to next cavity. Continue double
dilutions till the 12th cavity and discard the extra drop.

6. Add one drop of corresponding indicator cells, about 2%, in each cavity.
7. Thoroughly mix the contents and place at 40C for half an hour.

8. Then examine the contents for the presence of agglutination both macroscopically as
well as microscopically.

If the agglutination is present upto 4th cavity then the titre of the antiserum is 8;
up to 5th then is 16 and so on the basis of double dilutions.

4.6.4. Absorption Inhibition Method

1. Prepare the dilutions of antiserum of its penultimate titre (if the titre is 32 then prepare
a 16 dilution by taking one drop of the serum and adding 15 drops of normal saline to it).

2. Take a clean and dry cavity tile and mark three cavities as A, B and H and place one
drop of suitable dilution of anti-A serum, anti-B serum and anti-H lectin to the respective

3. Add one drop of the extract in each cavity, shake and keep at 4 0C for 2 hours.

4. Add one drop of 0.2% indicator cells in the respective cavities and keep at 4 0C for half
an hour.

5. Shake the tile and examine the contents for the presence of agglutination both
macroscopically and microscopically.

Agglutination in cavity Secretor status Blood group


+ + + Non secretor -

- + - Secretor A
+ - - Secretor B

+ + - Secretor O

- - - Secretor
Saliva and semen as forensic indicators
Both saliva and semen are products of exocrine glands (i.e. they
are secretions that are released to the outside of the body
through ducts) and both can provide valuable forensic evidence.
Saliva is a slightly alkaline secretion that is produced constantly
by the salivary glands and released into the mouth. Here it
lubricates the membrane surfaces and aids the chewing and
swallowing of food. Although it contains the enzyme salivary
amylase that begins the breakdown of starch, saliva does not have
an important role in the digestion of carbohydrates. In
addition, saliva also contains other enzymes, such as lysozyme,
some salts and mucin. The presence of saliva stains can be
demonstrated by detecting amylase activity. This is best done with
a specifi c assay method, such as ELISA (enzyme- linked
immunosorbent assay), that can distinguish salivary amylase from
other amylases, such as pancreatic amylase and bacterial amylase
(Quarino et al., 2005). Saliva is important forensically because
traces may be left at bite marks and in spit, from which it is
possible to isolate DNA. Furthermore, a number of drugs (in
particular those used illegally in sport) can be detected in saliva;
mouth swabs can be used as a source of DNA, thereby removing
the need for a suspect to provide blood for a DNA test.
Semen is gelatinous fl uid produced by the combined secretions of
the seminal vesicles, the seminiferous tubules, the prostate gland
and the bulbourethral gland. The typical ejaculate consists of
between 1.5 and 5 ml of semen and contains 40 – 250 million
spermatozoa. In addition to the sperm, the ejaculate also contains
the sugar fructose that serves as an energy store, citric acid,
calcium and a variety of proteins. Semen analysis is usually a
feature of cases of rape and sexual assault. Semen may also be
released following death and should therefore not be assumed to
denote recent sexual activity (Shepherd, 2003).
Both saliva and semen fl uoresce in UV light and spots observed on
the body or clothing are circled with an indelible marker for future
extraction and confi rmatory analysis. Commercial semen detection
kits are available over the Inter- net for spouses and partners who
suspect that their ‘ signifi cant- other ’ may be cheating on them.
These usually rely on detecting the enzyme acid phosphatase on
stained clothing and the assumption that a positive reaction can
only be explained by sperm belonging to another man (jealous
male) or consorting with another woman (jealous female). It
should be borne in mind that acid phosphatase is a very common
enzyme and the test is indicative rather than proof of the
presence of semen. Another, albeit more time – consuming, method
is to look for the spermatozoa. These may be found within the
vagina up to 5 days after sexual intercourse although the
frequency of detection declines rapidly with time (Willott and
Allard, 1982). Human spermatozoa are extremely small and soon
lose their tails, so a sensitive cytological test is required. The
most eff ective method is the ‘ Christmas tree test ’ (Allery et al.,
2001), which uses nuclear fast red and picroindigocarmine dyes to
stain the sperm red and green. More discriminative tests for
semen have been suggested, such as detecting the prostate
specifi c antigen (PSA) that is normally used in the diagnosis of
prostate cancer (Maher et al., 2002). Identifying an individual
from saliva or semen samples depends on either serological or
DNA profi ling. Approximately 75 – 85 per cent of the population are
termed ‘ secretors ’ , i.e. their body fl uids include the same or
similar profi le of antigens, antibodies and enzymes that are found
in their serum (although not the blood cells themselves, which are
normally confi ned to the blood vessels). This can therefore,
theoretically, enable an assailant to be identifi ed by sero- logical
profi ling from, say, a bite mark, spit or semen. However, the
serological profi le of the blood does not always match that of the
body fl uids and this reduces the eff ectiveness of this approach.
DNA profi ling off ers more powerful discrimination but even this
technique has its limitations. For example, in the UK, rapists are
now aware of the risk of detection by DNA profi ling and it is not
unusual for them to use spermicidal condoms. It has been
suggested that the use of such condoms (if found) might provide
an indication of when the assault occurred. In an experimental
study, the proportion of viable sperm within spermicidal condoms
was found to decline gradually from about 40 per cent to 6 per
cent over the course of 3 days (Gosline, 2005). This measurement
would be useful if there was a dispute about whether the condom
was ‘ planted ’ on the suspect or when the event took place.
Although these fi ndings are interesting, more work needs to be
done on how sperm viability within the condoms is aff ected by
temperature, light and other environmental conditions.
Furthermore, many rapists fail to ejaculate (Scutt, 1990) or if they
have had a vasectomy their semen lacks spermatozoa and hence
DNA. Despite the huge advances in forensic science, it is a sad
refl ection on justice in England and Wales that although the
number of reported cases of rape has risen, less than 6 per cent
of these result in a conviction (Home Offi ce fi gures, 2002,
1/hi/uk/ 4296433.stm). This emphasizes that the successful
resolution of a crime depends on more than the development of
ever more sophisticated laboratory techniques.

Faeces and urine as forensic indicators

Faeces and urine are sometimes found at a crime scene because
the culprit wishes to violate further the body or property of his
victim. Similarly, during a violent attack, both the assailant and
the victim can experience such stress and fear that defecation or
urination is involuntary. Faeces and urine may also be recovered
from a toilet that was not fl ushed and the body of a dead crime
victim. Faecal analysis can indicate a person ’ s diet and hence,
possibly, where he or she had
been eating. Some drugs and poisons can be detected in faeces
and it can also be a source of DNA (Hopwood et al., 1996). For
example, in 1995, the murderer of Monica Jepson, a 66-year- old
widow, left few clues behind at the nursing home in Birmingham
where she was staying. Just about all the police had to go on were
a partial fi ngerprint and some faeces left on the nursing home ’s
fi re escape. At the time, DNA profi ling techniques were still in
their infancy although it was possible to eliminate one suspect. By
2001, techniques had improved and a full profi le was obtained.
This was then compared to the National DNA Database© and a
match was obtained with John Cook. His DNA was present on the
database as a consequence of a previous off ence and when
brought in for questioning his fi ngerprints were found to match
those left at the crime scene. He was subsequently charged with
murder and sentenced to life in prison.
The study of DNA from faeces is diffi cult because it contains
substances that interfere with standard DNA extraction and
analytical techniques. For example, the human DNA needs to be
diff erentiated from the large amounts of bacterial DNA, whilst the
DNA amplifi cation process can be inhibited by the presence of bile
salts and plant polysaccharides (Lantz et al., 1997). However, a
commercially available kit for the extraction of DNA from faeces,
the QIAGEN QIAamp® DNA Stool Mini Kit, is now available and is
reportedly extremely eff ective (Vandenberg and van Oorschot,
2002; Johnson et al., 2005). Unless a person has a kidney or
urinary tract infection, urine does not normally contain many cells
although it is possible to extract DNA from urine and urine stains
(Nakazono et al., 2005). It is easier to obtain complete DNA
profi les from women ’ s urine than that of men because they shed
more epithelial cells (Prinz et al., 1993; Nakazono et al., 2005)
and also, presumably, because during the menstrual cycle their
urine will contain blood cells. In forensic science, urine analysis is
more frequently employed in the search for illegal drug use –
especially drug abuse in sport. An interesting piece of research
conducted by Zucatto et al. (2005) demonstrated the feasibility of
detecting cocaine and its main metabolites in sewage water. They
then scaled up their results to estimate the amount of the drugs
fl owing through the sewage water systems of four Italian cities.
Their fi ndings indicated a level of drug use far above that which
the authorities believed to occur. For example, they estimated
that the 5 million people living along the river Po were consuming
about 200 000 lines of cocaine every day. It is possible that a
similar approach might be used covertly to monitor levels of drug
use in a house, nightclub or prison by analyzing the drug
levels in the sewage pipes.