This document discusses various methods for blood group typing of dried bloodstains, including the Lattes test, absorption-elution assay, mixed agglutination technique, and absorption inhibition test. It also covers serological markers like enzymes, proteins, and HLA antigens that can be used for individualization in forensic analysis.

1. FORENSIC SEROLOGY

2. Reverse grouping of bloodstains: Lattes test LATTES
CRUST ASSAY PROCEDURE
1. Place small quantities of blood crust from a
specimen on a microscopic slide and place a cover
slide over the crusts. Prepare slides for A, B, and
O cells separately.
2. Prepare cell suspensions with saline (0.85%
NaCl in phosphate buffer, pH 7.4) for the A, B, and
O cells separately.
3. Apply a few drops of the A-cell suspension and
allow the cells to diffuse under the cover slip.
Repeat this step for B cells and O cells.
4. Incubate the slides in a moisture chamber at
room temperature for 2 h. 5. Examine results
under a microscope.

4. Forward grouping of bloodstains: Lattes test Absorption–Elution
Assay
 AEA is highly sensitive and can be used for testing
dried bloodstains. This method indirectly detects
the presence of antigens.
 The antigens are immobilized in a solid phase
 At low temperatures (4 degree overnight), the
antigens bind to their corresponding antibodies:
anti-A antibodies, anti-B antibodies, or anti-O
lectins.
 The excess unbound antibodies are removed by
washing, and the bound antibodies are then
eluted at higher temperatures (56degree for 30-
45 minutes)
 The eluted antibodies can then be identified by
an agglutination assay using A, B, and O indicator
cells.
 The bloodstains containing the A antigen can bind
to anti-A antibodies. The eluted anti-A antibody
can form agglutination with A cells. Likewise, for
type B blood and O blood group.

6. AN ALTERNATIVE TECHNIQUE FOR GROUPING OF BLOODSTAINS :Mixed
agglutination technique
 In this technique, the known antiserum (Anti-A sera Anti-B
sera Anti-H sera ) is added to the blood stain which has been
fixed to a surface such as the thread of a fiber. Incubated
overnight at 4 degree.
 After a period of absorption, the excess antiserum is washed
away with cold saline so that only the antibody, which had
reacted with the agglutinate, remains in the stain.
 Known indicator cells (0.5% suspension of A, B & O indicator
cells in isotonic saline) are then added. Place the slide in a
moisture chamber at 50 ⁰C for ten minutes.
 Red cells will attach themselves to the free ends of the bound
antibody.
 Remove the slide, allow it to cool, let sit at room temperature
for two hours and examine under a microscope.
 A positive reaction is indicated by the presence of cells, which
appear to be attached to the stain.

7. Secretors and Non-
Secretors
*mutation in FUT2 gene causes non secretor
status
FUT gene is located on chromosome 19

8.  This method involves the addition of tittered
antiserum (1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256)
to the stain (bodily secretions).
 The titer of the antiserum is adjusted so that most the
antibodies will be bound by the quantity of the stain
employed in the test if homologous antigen is present.
 Dry cavity tile mark A, B, H and place one drop of
suitable dilution of Anti A serum, Anti B serum, Anti H
serum.
 Add 1 drop of extract in cavity and incubate at 4
degree for 2 hours. If the corresponding antigen is
present in the stain, it will react with antibody and
decrease the titer of antiserum, so that it is no longer
available for agglutination of known test cells.
 Add one drop of 0.2% indicator cells and keep at 4
degree for 30 minutes.
 Thus the absence of agglutination of known test cells is
indicative of the presence of the antigen in the stain.
Absorption – inhibition test employed for ABO grouping of body
fluid stain.

9. SEROGENETIC MARKERS
Common Isoenzymes Used for Forensic Protein Profiling
Erythrocyte Isoenzyme Function Protein
Symbol
Number
of Alleles
Phosphoglucomutase transfers a phosphate group on an α-D-glucose monomer from
the 1 to the 6 positions in the forward direction or the 6 to the
1 position in the reverse direction.
PGM 2 (10
allele
detected
in IEF)
Erythrocyte acid
phosphatase
frees attached phosphoryl groups from other molecules during
digestion.
ACP/EAP 2
Esterase D splits esters into an acid and an alcohol in a chemical
reaction with water
ESD 2
Adenylate kinase catalyzes the interconversion of the various adenosine
phosphates (ATP, ADP, and AMP)
AK 2
Glyoxalase I catalyses the conversion of reactive, acyclic alpha-oxoaldehydes
into the corresponding alpha-hydroxyacids.
GLO 2
Adenosine deaminase needed for the breakdown of adenosine from food and for the
turnover of nucleic acids in tissues.
ADA 2

11. Serum Protein Polymorphisms
Serum Protein Function Chromosomal
location
Number of
alleles
Haptoglobin In blood plasma, haptoglobin binds with high
affinity to free hemoglobin released from
erythrocytes, and thereby inhibits its
deleterious oxidative activity.
Hp (16q22.1) 2
Group-specific component Human group-specific component (GC) is the
major vitamin D-binding protein in plasma.
Gc (4q12–13) 2
Transferrin Transferrin is a blood-plasma glycoprotein,
which plays a central role in iron metabolism
and is responsible for ferric-ion delivery.
Tf (3q22.1) 3
Protease inhibitor (α1-
antitrypsin)
Alpha-antitrypsin (AAT) is a serine protease
inhibitor, which inhibits the proteolytic enzyme
elastase (it is an enzyme from the class of
proteases that break down proteins)
Pi (14q32.1) Many

12. HLA antigens and HLA typing
 HLA (human leukocyte antigen) is
the human major
histocompatibility complex
(MHC), a multigene family involved
in the defense of humans (and all
vertebrae) against pathogens.
 Plays vital role in cell mediated
immunity.
 The classical loci, routinely studied
in human medicine, are HLA-A,
HLA-B, and HLA-C for class I, and
HLA-DRB1, HLA-DQB1, and HLA-
DPB1 for class II.

13. HLA class I HLA class I I
polymorphic α-chain
combined with a
monomorphic β-
globulin chain
two polymorphic
chains (α and β)
Expressed on almost
all nucleated cells.
Expressed
constitutively only on
macrophages,
dendritic cells, and B
cells, as well as
activated T cells and
thymic epithelial cells
present endogenously
processed peptides to
CD8+ T
present peptides to
CD4+ T cells.

14. HLA TYPING
Sequence-specific oligonucleotide probe (SSOP)
hybridization
Sequence-specific primers (SSP)
Sequencing-based typing (SBT)
Next generation sequencing (NGS)
DNA Typing Methods: Most HLA typing methods in use are
based on amplification of specific HLA gene portions from
genomic DNA using polymerase chain reaction (PCR).