COOMB'S TEST.pptx

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. Coombs test 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. Rh Incompatibility 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. Coomb’s reagent 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. Types of Coombs Test

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

7. Steps Prepare normal saline-cell suspension (5%) Take a test tube 950 µ L Normal saline + 50 µ L 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)

8. 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

9. 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

10. Also it can be tested by AHG GEL CARD

11. Principle of GEL card method 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 µ L) into card then centrifuged the card incubate it for 30 mint on water bath Observe the results

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

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

14. 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 µ L NS + 50 µ L Washed cells (5% Cell suspension)

15. 50 µ L from Cell suspension add on all three tubes + + + 1 drop Anti D 50 µ L NS Patient 50 µ L 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

16. + - T Pooled O+ve blood

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