AHG TESTS.ppt MLS KMTC anti human globulin tests

Anti human globulin tests

1. ANTIGLOBULIN TESTS

2. By
Adnan Hussein
Lecturer
Transfusion Science

3. Antiglobulin Tests
Preparation Of AHG
1.Purified human globulin is injected into the
rabbit.
2.After immunization, the rabbit will produce an
antiserum with antibodies against human
globulin
3.Human beta globulin and gamma globulin is
used as a source of antigen

4. 1.When human gamma globulin is used
alone to produce antihuman globulin, the
AHG formed is referred as monospecific
AHG- Monoclonal AHG
2.When both human gamma globulin and
beta globulin are used to produce AHG it
is referred to as polyspecific AHG-
Polyclonal AHG

5. 3.Monospecific AHG detects gamma globulins while
polyclonal AHG detects both gamma globulin and
Complements.

4.After a suitable cause of immunization, the rabbit is
bled using sterile apparatus.
5.Blood is collected in a plain container to allow it to
clot
6.Separate the clear serum into another container.

6. 7.The serum is now absorbed to remove
unwanted antibodies
8.Standardized ie adjusted to the required
titre.
9.Coloured and sterilized by filtration.
10. It is then labelled
11.Stored at 2
0
C or - 20
0
C

7. DIRECT ANTIGLOBULIN TEST
•Also referred to as Direct Coombs Test-
DCT

Purpose
•Performed to detect red cells which are
sensitized in vivo (while in the circulation )
with IgG incomplete antibodies

8. •Direct Coombs test is of great value in the
diagnosis of;
1. Haemolytic Disease of the Newborn
2. Incompatible transfusion reaction
3.Autoimmune haemolytic anaemia

9. Procedure
1. Make a 2-5% cell suspension
2. Label a tube as DCT
3. Put 2 drops of cell suspension to the tube
4. Add a drop of AHG serum and mix
5. Centrifuge at 1000 rpm for 1 min.
6. Examine for agglutination Macoscopically /
Microscopically

10. Results

•Agglutination is reported as DCT positive
•No agglutination is reported as DCT negative

Interpretation
•DCT positive indicates that the antibodies have sensitized
cells in vivo therefore presence of incomplete antibody

11. •DCT negative indicates that no antibodies
had sensitized the cells in therefore
absence of incomplete antibody

12. • Results

•Agglutination is reported as DCT positive
•No agglutination is reported as DCT negative
• Interpretation

•DCT positive indicates that the antibodies have sensitized
cells in vivo therefore presence of incomplete antibody

13. •DCT negative indicates that no antibodies
had sensitized the cells in therefore
absence of incomplete antibody

14. •INDIRECT COOMBS TEST
•Also called Indirect Coombs Test ( ICT )
•Purpose
•Performed to detect sensitized cells in
vitro (outside the patient’s circulation)

15. •Importance;
a)Detection and identification of irregular
antibodies
b)Detection of incompatiblility-crossmatch
c)Detection of antigens not demonstrable by
the normal method eg the D
U
antigen-low
grade D
U

16. •Procedure
1.Make a 2-5% cell suspension of pooled
O+ve cells
2.Put 2 drops of the patient’s serum into a
tube
3.Add a drop of O +ve cell suspension and
mix
4.Incubate at 37
0
C water bath for 30 min

17. 5.After incubation , was the contents of
tube at least 3 times in normal saline
6.Discard the supernatant completely after
the last wash
7.Add a drop of AHG serum and mix
8.Centrifuge at1000 rpm for 1 min
9.Examine for agglutination
macroscopically and microscopically

18. Results

•Agglutination is reported as Indirect
Coombs’ positive
• No agglutination is reported as Indirect
Coomb’s negative

19. •Interpretation
• ICT positive indicates that the cells have been sensitized
in vitro therefore presence of incomplete antibody


• ICT negative indicates that no sensitization therefore
absence of incomplete antibody

20. •Note
•Positive and Negative controls should be
used
•+ve control- Use of O positive sensitized
with incomplete antibody
•-ve control- Use of non sensitized cells

21. •Preparation of positive control cells
1.Dilute 1 in 10 of commercially prepared
anti D ie 1 drop anti D with 9 drops of
normal saline.
2.Wash O +ve cells
3.Mix equal volumes of cells and the diluted
anti D
4.Incubate at 37
0
C for 30 min

22. 5.Was thoroughly in saline and discard the
supernatant saline
6.Prepare a cell suspension of the
sensitized cells

23. •Causes Of False Results
•False positive
1.Bacterial contamination of reagent or
serum
2.Inadequately absorbed AHG will cross
react with other antigens

24. •False negative
1.Inadequate washing of cells will neutralize
the coomb’s serum
2.Failure to add the Coomb’s reagent
3.Impotent AHG serum due to improper
storage ,contamination of apparatus,
saline which will neutralize the AHG

25. Note
•As all human serum contain globulin ,it is
essential that any globulin not attached to the
cells be removed by washing in large volumes of
saline before the addition of AHG serum
•If they are not removed , the Coomb’s serum will
be neutralized by the free globulins and false
negative reaction will be obtained
•Use of controls is therefore very important which
will check the potency of AHG

26. ANTIBODY SCREENING TEST
•Purpose To detect incomplete antibodies
(irregular antibodies ) in vitro

Incomplete antibodies may arise from;
1. Isoimmunization of pregnancy
2. Previous blood transfusion
3. May occur in absence of known antigenic
stimulation eg autoimmune disease

27. There presence may be indicated by;
1.A difference in the results of ABO cell and
serum grouping
2.Incompatible Cross match
3.A blood transfusion reaction
4.A positive test for irregular antibodies

28. Procedure
1.Label 6 tubes as SRT, S37
0
C , A37
0
C , C37
0

C , S 4
0
C and E 37
0
C
2.Put 2 drops of the test serum into all tubes
3.Add 2 drops of pooled O positive cell to all the
tube except Enzyme at 37 0 C,
4.Add a drop bovine albumin to the tube A37
0
C ,
and mix

29. 5.Add a 2 drops of enzyme treated cells to the tube 37
0
C
and mix
6.Incubate the tubes at their respective temperature for 1
hour
7.Wash contents of tube C37
0
C , 3 times in saline and
remove supernatant saline completely
8.Add a drop of AHG to C37
0
C and mix
9.Centrifuge all the tubes at 1000 rpm for 1 min
10.Examine for agglutination macroscopically and
microscopically

30. Results
•Agglutination in any of the tube reported as AST positive

•No agglutination in all the tubes is reported is reported as
AST negative
Interpretation
•AST positive means presence of incomplete antibody .

AST negative
means absence of incomplete antibody .

31. •AST positive means that the serum has an
irregular (incomplete ) antibody which react
at A 37
0
C and C37
0
C


Saline
4
0
C
SRT S 37C A 37C C37 C E37 CComment
s
-ve-ve-ve-ve-ve-veAST -ve

32. ANTIBODY IDENTIFICATION TEST
Purpose
• To identify an offending antibody ie the antibody
presenting the problem and causing the positive
reaction in antibody screening above

•In this test ,panel cells ( identogens) are used

33. Procedure
1.Label the test tubes according to the way the
panel cells are labeled eg 1-10 or A-P
2.Put 2 drops of the test serum into all tubes
3.Add a drop of panel cells to the corresponding
tubes eg panel A to tube A
4.Mix the contents in each tube
5.Incubate the tubes at the temperature at
which the antibody reacted 1- 2 hrs

34. 6.Centrifuge all the tubes at 1000 rpm for 1
min.
7.Observe for agglutination both
macroscopically and microscopically
8.Note the tube number which reacts and
check what antigen on the panel cells
9.The antigen will enable you to know the
antibody that was present in the serum

35. Antibody Titration Test
•Involves making serial dilutions to
determine the strength (Titre) of an
antibody against the corresponding
antigen

36. Purpose
1.To determine the titres of antibodies A and B
in serum.
2.In testing the potency of an antiserum for use
in ABO cell grouping
3.Screening O negative blood for high titre anti
A and anti B to determine safe universal
donors

37. Procedure

1.Label 10 tubes as 1:2, 1:4, 1:8, 1:16, 1:32,
1:64 up to 1:512
2.Put 3 drops of saline into all tubes
3.Add 3 drops of the test serum to the first tube
only.
4.Mix the contents in the first tube and transfer 3
drops to the second tube

38. 5.Mix the contents in the 2
nd
tube and
transfer 3 drops to the 3
rd
tube
6.Continue with the process until the last
tube
7.Add 2 drops of the 5 % cell suspension of
known cells to all the tubes and mix
8.Centrifuge all the tubes at 1000 rpm for 1
min

39. 9.Examine for agglutination
macroscopically and microscopically

40. Results
1:21:41:81:161:321:641:1281:2561:512
+ve+ve+ve+ve+ve-ve-ve+ve+ve