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Immunology in Forensic Science
Immunology in Forensic Science
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?
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
Director, State Forensic Science Laboratory, Himachal Pradesh, Junga-173216. (India)
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of some immunological methods used in Forensic Science is given in the
succeeding paragraphs of this chapter.
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.
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.
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.
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.
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Immunoblotting
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.
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2. The Cellular 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
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
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Rh System
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.
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%
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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.
It was discovered by Cutbush and Chanarin in 1950 (Boorman and Dodd, 1957).
Phenotype as defined Genotype Frequency %
by Anti Fya and Fyb
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 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
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 Lub 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.
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Phenotypes Genotype Grequency %
Lu (a+b+) Lua Lub 8%
Lu (a+b-) Lua Lua 0.2%
Lu (a-b+) Lub Lub 92%
Lu3, Lu4 and Lu5 antibodies have been discovered later in this system.
P System
P2 21%
Pk1 Very rare
Pk2 Very rare
p Very rare
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.
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 stain’s testing. The HLA antigens at A&B locus
have been tried for testing various stains.
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Application of rare blood groups in Forensic Science
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.
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.
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