This document discusses several methods for determining blood types from bloodstains, including the Lattes test, absorption-elution assay, mixed agglutination technique, and absorption-inhibition test. The Lattes test involves incubating blood crusts with cell suspensions to look for agglutination under a microscope. The absorption-elution assay detects antigens in dried bloodstains by binding antibodies at low temperatures, then eluting and identifying the antibodies. The mixed agglutination technique uses known antiserum and indicator cells to look for cell attachment to stains. The absorption-inhibition test employs titered antiserum and measures its ability to agglutinate cells after exposure to a stain, indicating the presence

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.