Immunology In Forensic Science

Dr. J.R. Gaur

Introduction
The basic task of the forensic serologist and immunologist is to answer certain
specific questions related to the examination of blood, other body fluids and their
stains. Sometimes, other tissue materials like bones, hair, skin and flesh etc. are
also required to be examined from forensic aspects. The major questions, which
have to be answered are usually:

Is the stain of blood or of a specific body fluid like saliva, semen or vaginal
secretion etc. ?
If it is of blood, semen or saliva stain, is it of a human being. ?
If it is of human origin, to which group or type it belongs. ?
Is it possible to obtain further information towards individualization of stains
or biological materials sent for examination?

Such questions a forensic serologist has to answer after forensic, Chemical,

Biological, Serological and immunological laboratory investigations in every case
of murder, assault, rape, dacoit or hit and run accident etc., where there has been
bloodshed or some exhibits stained with blood or body fluids have been
recovered by the police during investigation of different cases and are sent to a
forensic laboratory for examinations.
Sometimes, the evidence of body fluids typing is an excellent corroborative
evidence to link or de-link the accused and the weapon of offence with the crime.
In some cases, where there are no eye-witnesses, the blood grouping or body fluid
typing evidence may be concluding physical evidence in favour of , or against the
accused.

The aforesaid four questions can be answered by the forensic serologist after
performing chemical, microscopic, immunological, enzyme and serum protein
typing and DNA profiling tests. However, after the discovery of ABO blood
group system by Land Steiner, K. (1901), there has been almost an invasion in the
twentieth century for introducing immunological methods in Forensic Science.
These methods are still of utmost use in the laboratories devoid of DNA typing
facilities or in routine tests. Immunological methods are prevalent in forensic
science even today as these tests are very sensitive and specific. A brief account
of some immunological methods used in Forensic Science is given in the
succeeding paragraphs of this chapter.
Director, State Forensic Science Laboratory, Himachal Pradesh, Junga-173216. (India)

What is Immunology?
Immunology is the study of antigen and antibody reactions and the immune
response produced by the antigens in a living being. Antigens are the substances,
which ilicit an immune response in the host. Antigen may be of several types,
soluble and particulate proteins, viruses, sub-cellular particulates and entire
complex cells such as tumor cells and bacteria, which are foreign to the host.

Species of Origin test

After the identification of biological stains and other tissue material, a forensic
scientist has to find out species of origin of the same, which can be done by the
various methods through immunological antigen and antibody reactions. The
stain or tissue material in the test provide antigenic material for the purpose and
species specific antibodies raised are used as antisera in these tests. The test can
be performed as ring test in the precipitin tubes or by immuno diffusion in Agar
or Agarose gel. Further this test can be carried out by cross-over electrophoresis,
immuno electrophoresis and rocket immuno electrophoresis etc. The appearance
of milky bands and at the meeting junction of antigen and antibody due to the
formation of antigen and antibody complexes indicative of positive reaction. In
1946 Qudin developed the single tube diffusion method and in 1949 Ouchterlony
prescribed the method of antigen antibody reactions for the detection of origin of
species in gels. Various methods were devised by the scientists in the succeeding
years.

Raising of specific species

Strict immunization schedule is a must for good titre of antisera. The amount of
antigen, type of animal and the route of antigen administration play a vital role in
the immune response. Minute amounts of antigen is desirable for immunization.
The larger doses of antigen induce tolerance and hence are not advisable. Rabbit
is the convenient and ideal animal for the experimental immunization. Best route
of administration for particulate antigens is peritoneal and for the soluble antigens
the intradermal or intramuscular route is recommended.

Hapten conjugation to carrier macromolecules

Haptens are low weight molecules, which cannot produce an immune response.
These smaller molecules have to be conjugated to a carried molecule, usually a
protein like Bovine Serum Albumin before they are used as immunogens for the
production of antisera. Several compounds in forensic investigations, which fall
in the category of haptens.

Immunoblotting
One of the techniques largely used to detect antigen-antibody reactions is
Immunoblotting technique. This involved immobilization of the antigen on a
matrix such as nitrocellulose (NC) paper and probing with an antibody.
Immunoblotting enhances the detection of antigens because, the antigens after
they are transferred from the gel into the NC membranes, they are readily
available for the antibody probes for their detection. Antigens, after their transfer
can be stored for longer time at 200 C for future use. The visualization of antigen
and antibody complex is accomplished by using a second-antibody specific to the
first antibody. The second antibody is radioactively labeled or coupled to enzyme
like horse-radish peroxidase. The latter kind uses a colour-reaction for
visualization. In the Immunoblotting technique the conditions are provided such
that only the specific experimental conditions are such that only interactions of
high specificity are retained before visualization, Kashyap (1989).

Microbead Based ELISA for the quantification of L-feto

protein (AFT)
In 1956 Bergstrand discovered L-fetoprotein (AFP) for the first time in human
foetal serum. AFP is a single polypeptide with a molecular weight of
approximately 70,000 da. The physicochemical properties and aminoacid
composition of AFP are similar to those of albumin. It is secreted into the foetal
serum and reaches a peak level at about 13 weeks of gestation period and
gradually declines thereafter. From forensic point of view AFP is useful for
discriminating the foetal blood stains from the adult blood stains. It can be done
with microbead based ELISA technique.

Blood Groups
Karl Landsteiner on the basis of his observations on blood classified human blood
into four groups A, B, AB, and O in 1901 and discovered ABO blood group
system which has been widely in use for blood transfusions, population genetics
studies and Forensic Analysis through out the world.
Since the discovery of ABO blood group system by Karl Landsteiner, the
knowledge in Forensic Serology has expanded tremendously. Today, more than
160 antigens, 150 serum proteins and 250 cellular enzymes have been found in
human blood. Three classes of blood constituents have been chosen by serologists
for the analysis of blood samples.
1. The Group specific antigens.
2. The Cellular enzymes and proteins.

3. The serum enzymes and proteins.

The basic ABO blood group system is polymorphic system in which several other
rare forms of A, B & H antigens have been added up later on and are very much
useful in forensic sciences for the establishment of identity of a person.

M N System
It is a two allele system discovered by Landsteiner and Levine in 1927. They
called the antigens of this system to be M and N and alleles as L M and L N . Later
on like ABO several subgroups of M and N were discovered. Among these is one
consisting of persons who have the rare, weak antigen N2 , which depends on the
presence of the allele LN2 another of those who have the antigen M g , transmitted
by the very rare allele designated as L mg. The antigen M g is remarkable in that it
does not react with either antibody anti M or anti N. Only heterozygous L Mg
L M and L Mg L N individuals have been found so far. A homozygous L Mg L Mg
person who must be excessively rare-tested with anti M and anti N sera
would be neither M nor N nor MN. Distribution of MN types in Europeans is
M=28%, N=22% and MN=50%.

The S s Antigens
In 1947, twenty years after the discovery of M and N antigens, a new antigens, S,
was found. Later on recessive antigen s was discovered and the genotypes, SS,
Ss and ss were also recognised. These antigens S and s were found associated
with all MN types. Percentage of Ss types in Europeans is SS=11%, Ss=44% and
ss=45%.

Lweis Types
Mourant in 1946 found that an unknown antibody believed to be naturally
occurring was found in the blood of two different persons. This antibody
recognised an antigen present in about 22% of the English population. The
system was called as Lewis after one of the two individuals in which it was
found. Later on Le positive to be secretors of ABH substances in saliva. Common
phenotypes of Le (a+b-) were found to occure in most of the world populations.
The rare phenotype Le (a+b+) is found in infants but only upto the age of one
year or so, it is however very rare in adults. Le (a-b-) very uncommon in Indian
populations is as high as 46% in West African Negros. Le x antibody is found to
react with Le (a-b-) blood samples. Two more antigens Lec and Led which are very
rare have also been identified in some human populations.

Rh System
4

Levine and Stetson (1939) had described an atypical antibody which later was
shown to be anti Rh in specificity. The real importance of Rh factor was however
realized in 1940 when Weiner and Peters reported even after ABO compatibility
in transfusions sera of some persons showed still cross reactivity. Levine and his
co-workers in 1941 published series of papers when they found that Rh negative
mother got immunized from a Rh+ve foetus which gave rise to erythroblastosis
foetalis or HDNB.
The eight main Rh haplotypes had been identified.
-------------------------------------------------------------------------------------------------Fisher Notation
Weiner Notation
Frequency in English
Populations.
-------------------------------------------------------------------------------------------------CDe
R1
0.421
cDE

R2

0.141

cDe

Ro

0.026

CDE

Rz

0.002

Cde

0.010

cdE

0.012

CdE

ry

Very low

cde

0.389

Rh system is further complicated by Cw Cx Cv Cu g, v and Du etc. genes. Which are

quite rare but Du is common than the other and is a Negroid trait.

The Kell system

This antibody was found in the serum of a mother whose infant suffered from the
haemolytic disease of the new born (HDNB). K antigen was found to occur in
10% of the British population and it was postulated that the system was governed
by a pair of allelic genes called K and k which controlled the production of
corresponding antigens K and k. The groups being:
Phenotype

Genotype

Kell Positive

KK= 0.2%
Kk = 10.0%

Kell Negative

kk = 90.0%

Later on k gene was called to be cellano factor and another rare type in this
system, i.e. K k or K0 k0 was discovered. Till today 21 Kell related antigens
have been described, but all have not been found to be Kell alleles.

The Duffy System

It was discovered by Cutbush and Chanarin in 1950 (Boorman and Dodd, 1957).
Phenotype as defined
by Anti Fya and Fyb

Genotype

Frequency %

Fy (a+b-)

Fya Fya

17

Fy (a+b+)

Fya Fyb

Fy (a-b+)

Fyb Fyb

49
34

Later on, three antigens Fy3 Fy4 and Fy5 were discovered and added to this system
which were rare in nature.

The Kidd blood group system

The Kidd blood group system was discovered in 1951, the antibody being found
in the serum of woman, Mrs. Kidd, whose sixth child suffered from haemolytic
disease of the newborn. The role of the antibody in connection with this disease
could not be assessed because the naterual serum also contained Anti-Kell. The
Kidd antibody has been found to be stimulated either by transfusion or by
pregnancy or both. The antibody was called anti-Jk later anit-Jk was discovered
Phenotype

Genotype

Approx. Occurrence

Jk (a+b-)

Jka Jka

26%

Jk (a+b+)

Jka Jkb

50%

Jk (a-b+)

Jkb Jkb

24%

This system has not further been expanded so far.

Lutheran System
In 1946 it was found that an unknown antibody in the blood of a patient who had
received many blood transfusions. The antibody was immune, the Lu b was
discovered by Cutbush and Chanarin in 1956 in Origin of Man (Buettner and
Janusch, 1966). None of the antibodies of this system had any clinical
significance.

Phenotypes

Genotype

Lu (a+b+)

Lua Lub

Lu (a+b-)

Lua Lua

Lu (a-b+)

Lub Lub

Grequency %
8%
0.2%
92%

Lu3, Lu4 and Lu5 antibodies have been discovered later in this system.

P System
System discovered in 1927, however in 1949 anti - P was discovered by making it
active at 40 C.
Phenotype

European frequency

P1

79%

P2

21%

Pk1

Very rare

Pk2

Very rare

Very rare

The I blood Group System

In 1956 one new antibody was found in the serum of a patient suffering from
haemolytic anemia of the cold antibody type, it was called-I. Of 22000 donors
tested, only five were negative and were therefore designated as i falls. Later, I1
was found only in whites and i2 in Negros.
Further several other blood group systems were added in Serology viz. SID,
Wright (Wr), Dombrock (DO), Diego (Di) and Colton etc. But the antigens are
not very stable in stains and thus are of forensic significance only in paternity
disputes if the corresponding antisera are available. Detection of these antigens in
stains is not possible as the antigens being weak.

The HLA System

Highly polymorphic system is of utmost help in Forensic Science for the typing
of blood samples in crime cases and paternity disputes. Though, there has been
problem of cross reactivity in stains testing. The HLA antigens at A&B locus
have been tried for testing various stains.

Application of rare blood groups in Forensic Science

Rare antigens whenever detected in blood or blood stains are of utmost
importance and evidential value in the individualization of blood and in paternity
disputes. The HLA antigens make HLA systems very much polymorphic and
helped a lot in organ transplants and also solving paternity disputes. However, the
probability of matching on the basis of all blood group systems Serum proteins &
enzymes never reaches beyond 94 to 95%. But the rare blood group antigens have
always been of utmost value, whenever, found as it brings the probability nearing
100%.

Limitations of all rare blood groups in Forensic analysis.

All rare blood groups systems had not been of Forensic utility when the tests have
to be carried from the blood stains and other body fluid stains. Because the
putrefication at high temperature and humidity and with the elapse of time in
reaching to the experts for examination in the Laboratory. Gene frequency of
some rare antigens and there detectibility periods after preserving blood stains at
room temperature and deep frozen are presented through the transparences which
indicate that the maximum detectable period of the MN antigens to be six months
and Lewis antigens up to three days only. And other are antigens which are
sometimes less resistant to the high temperature and humidity cannot be detected
from the stains.

Identification of Species of Origin and grouping tests in

Forensic analysis.
Scientist all over the world have worked extensively for devising various methods
of analysis and modifying them extensively in the 20 th Century. The readers are
referred to the important compilations like Biology Methods Manual (1978) of
Metropolitan Police, Forensic Science Laboratory, London and the works of
Chowdhuri, S. (1979), Kashyap, V.K. (1989) and Gaur, J.R. (1989) in India, who
have presented important methodology and data on the subject. It may also be
mentioned that Gaur, J.R. and Bhalla, V. (1993) presented A serological profile
of the people of Haryana (India). The blood group genotype and phenotype
frequency data contained in this study gives recent data of blood groups in that
part of India and can be used for forensic purposes in the Indian contest. Gaur,
J.R. (1989) also studied and presented data on the detectibility studies of various
blood group antigens in the environmental conditions of Haryana and in
controlled laboratory conditions.
Thus, immunological methods are of utmost importance in Forensic Serology.
They have played a significant role in crime investigation and the administration
of Justice in the past and present and shall continue to do so in future.
8

References:1. Bergstrand, C.G., (1956), Demonstration of new protein fraction in serum from
human fetus, Scand J. Clin & Lab. Invest., 8-174.
2. Biology Methods Manual , (1978), Metropolitan Police Forensic Science
Laboratory 109, Lambeth Road, London SE 1 7 LP, England.
3. Boorman, K.E. and B.E. Dood, (1957), Blood group serology., Little, Brown,
Boston.
4. Buetner, J. and Janusch, (1969), Origins of man, Wiley Eastern Private Limited.,
ND (India), p.p 465-495.
5. Chowdhuri., S., (1979), Examination of biological stains, BPR&D, ND, p.p. 1-51.
6. Gaur, J.R., (1989), A study of blood group polymorphisms in selected populations
of Haryana with special reference to their detectibility in dried blood on ageing,
Ph.D. thesis, Punjab University, Chandigarh (India), p.p. 1-191 (Unpublished)
7. Gaur, J.R. and Bhalla, V. (1993), A serological profile of the people of Haryana in
people of India, editors: S.B. Roy and A.K. Ghosh, Inter-India Publications,
New Delhi, p.p. 70-83.
8. Kashyap, V.K., (1989), Immunological methods in Forensic investigation
(protocols), CFSL, MHA, GOI, BPR&D, Hydrabad, 1-16.13.
9. Landsteinder, K. (1901), Uber Agglutination serscheinungen normalen
menschlichen. Blutes. Wein. Klin. Wschr., 14, 1132-1134.
10. Landsteinder, K. and P. Levine, (1927), A new agglutinable factor differentiating
individual, 495.
11. Mourant., A.E., (1954), The distribution of human blood groups, Black Well,
Oxford, London.
12. Ouchterlony, (1949), Antigen antibody reactions in gels, Acta Pathol.
Microbiol. Scand., 507.
13. Qudin, (1946) Methods d analyse immunochimique par precipitation specifique
en milieu gelifie C.R. Acad. Sci., 222
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Scientific Publishers.