coombs test, introduction with principle and whole laboratory procedure, also u will read about ,how to perform direct and indirect coombs test? and how to report them?

1. COOMBS TEST
OR
ANTIGLOBULIN
TEST
Prepared By-
ARPITA CHANDRA

2.  An immunological test
 To detect the presence of antibodies against the red blood
cells.
 To diagnose Rh incompatibility by detecting Rh antibody from
mothers and baby serum.

3.  When Rh negative Mother delivers a Rh positive
baby (Rh Ag +ve) , during birth, some Rh +ve RBCs
may pass from fetus to the maternal circulation and
may induce Rh Ab formation in mother
 In future these Rh antibodies effect Rh positive
pregnancies.(cross the placenta)
 Causes hemolytic diseases
Erythroblastosis Featalis

4.  Antiglobulin - Anti IgG
 Made by immunized rabbits with human gamma
globulin
 Bind to the Fc portion of Rh antibody bound on
RBCs, resulting in visible agglutination.

5. Direct Coombs test- it detects Rh
antibodies bound to RBCs in vivo present
in baby’s serum by directly adding
commbs reagent.
Indirect Coombs test- It detects free Rh
antibodies present in maternal serum after
the first delivery of Rh positive baby.

7. Procedure
Requirement-
 Normal saline
 Blood Sample (EDTA)
 Centrifuge
 Pipette
 3 Test tube

8. Prepare normal saline-cell suspension (5%)
Take a test tube
950 µL Normal saline + 50µL sample
Mix it
Take another tube- Add 1-2 drop of suspension + Normal saline
Centrifuged it for 1min at 1000rpm
Discard supernatant
wash x3 (to remove unbound globulins)

9. A test tube- Add 2-3 drops of washed RBCs
+
Anti-Human sera (Coomb’s reagent)
Incubate it 30 min
after incubation
Observe for Agglutination
Macroscopically and Microscopically

10. Take one drop of RBCs on a glass slide
place a coverslip and observe clumping
of RBCs under microscope
Clumping of RBCs –
Incompatible(coomb’s test positive)
Scattered RBCs seen(Compatible)
- Negative

11. AHG

12. The gel column acts as a filter that traps agglutinated RBCs (if there
is Ag- Ab complex)
but they pass through the gel column during the centrifugation
based on size (If there is no Ag- Ab complex)
Procedure
Add Patients washed RBCs cell suspension (50 µL) into card
then centrifuged the card
incubate it for 30 mint on water bath
Observe the results

13. RBCs settle down on bottom of the well- Compatible
RBCs on the surface of the gel
- Incompatible
MF = Mixed field agglutination

15. + - T
Requirements-
Normal saline
 Patients serum (mother)
 Centrifuge
 Pipette
 5 Test tubes
 O+ pooled cells (Rh +ve)
 Anti- D Reagent (Anti D IgG)

16. Steps
Preparation of O+ Pooled cells- by adding (three blood samples
of o+ ve blood group)
make cell suspension of O+ve blood
1. Take 2-3 drops of RBCs + 2/3 Normal saline
2. x3 wash RBCs, centrifuge(1000rpm, 2min)
discard supernatant
3. A tube- Add 950 µL NS + 50 µL Washed cells
(5% Cell suspension)

17. 50 µL from Cell suspension add on all three tubes
+ + +
1 drop Anti D 50 µL NS Patient 50 µL serum
Incubate it 1 hr at 37C then wash x3 with NS
Add Anti- Sera on Tube(T)
Incubate 5 mint
Observe Macro / Microscopically
+ - T

18. +
- T
Pooled
O+ve
blood

19. Results
If Aggluitination seen at the bottom of Test tube (Positive ICT)
No agglutination Seen (Negative ICT).