BB LABORATORY PROCEDURES IAT (INDIRECT ANTIHUMAN GLOBULIN TEST)

DAT (DIRECT ANTIHUMAN GLOBULIN TEST) TUBE METHOD

TUBE METHOD Specimen:
Specimen:  EDTA-anticoagulated WB (5% RBC suspension)
 EDTA-anticoagulated WB (5% RBC suspension) Reagents:
Reagents:  AHG reagent: anti-IgG
 AHG reagent: anti-IgG  Group O antibody detection cells: donor-pooled group O, px- unpooled cells
 (-) control reagent: 6% bovine albumin or serum  Check cells: IgG-coated group O red cells
 Check cells: IgG-coated group O red cells  0.85%-0.90% NSS
 0.85%-0.90% NSS  Incubator/ water bath: 37°C
Procedures: Procedures:
1. Label 2 test tubes: Px X and Control
2. Dispense 1 drop of 5% RBC suspension into each labeled test tube IMMEDIATE SPIN PHASE
3. Wash the contents each tube 3 times (3-5 drops) with NSS (2/3 of the amount). Remove the 1. Add 2 drops of serum into a labeled test tube (px Y)
supernatant 2. Add 1 drop of 5% group O red cells into the labeled tube. Cover with parafilm and gently mix.
4. Add 1 drop of AHG reagent and mix 3. Centrifuge at 3400 rpm for 10-15 sec (1 min)
5. Cover the test tube with parafilm and centrifuge at 3000 rpm for 10 sec (1 min) 4. Resuspend.
6. Resuspend. 5. Grade the result
7. Grade the agglutination
8. If initial centrifugation is NON reactive, incubate or allow the tubes to stand for 5 minutes at * IF NO AGGLUTINATION, PROCEED TO THE NEXT STEP *
room temperature (20°C), centrifuge and read again.
9. Confirm negative results by adding check cells 37°C INCUBATION PHASE
10. Centrifuge again for 1 min 1. Add 1 drop of 22% bovine serum albumin into the previous nonreactive tube in immediate spin
11. Examine cells for agglutination 2. Cover with parafilm and gently mix
Interpretation: 3. Incubate at 37°C for 10-15 minutes: waterbath
 (+) DAT when agglutination is observed either: 4. Centrifuge at 3400 rpm for 10-15 sec (1 min)
- immediate centrifugation 5. Resuspend
- after centrifugation following incubation at room temp 6. Grade the result
 (-) DAT when no agglutination is observed.
- to confirm for true negative, add check cells. * IF NO AGGLUTINATION, PROCEED TO THE NEXT STEP *
- True negative = (+) agglutination after adding check cells
 INTERPRETATION: There is/no in vivo RBC sensitization AHG PHASE
Grading: 1. Wash the tubes 3 times with NSS (2/3 of the amount or 5-10 drops). Remove the supernatant.
2. Add 1 drop of AHG reagent
Grading of Tube Agglutination Reactions 3. Cover with parafilm and gently mix
4. Grade the result
Macroscopic (Visual Readings):
5. If (-), add check cells to confirm for its true negativity
Grade Description of Reaction
- True negative = (+) agglutination after adding check cells
4+ ONE solid agglutinate; CLEAR background
6. Centrifuge at 3400 rpm for 1 min.
3+ SEVERAL LARGE agglutinates, CLEAR background 7. Examine cells for agglutination
2+ MEDIUM-sized agglutinates, CLEAR Interpretation:
1+ MANY SMALL agglutinates; TURBID background  At any phase, if there is an agglutination, test is (+) at that specific phase and no need to
0 NO agglutination; TURBID background proceed to another phase
Microscopic Readings  If there is no agglutination, at any phase, proceed to the following phase until AHG phase. Add
Grade Description of Reaction check cells if the result is still not reactive.
W+ BARELY visible agglutination  INTERPRETATION: There is/no in vitro RBC sensitization
0 NO agglutination; cells float FREELY

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 ABO BLOOD TYPING and Rh TYPING CROSSMATCHING (MAJOR)
FORWARD GROUPING (red cell or front typing) TUBE METHOD
Specimen: Specimen:
 EDTA-anticoagulated WB (5% RBC suspension)  EDTA-anticoagulated WB (5% RBC suspension)
Reagents: Reagents:
 Anti-A (blue)  0.85%-0.90% NSS
 Anti-B (yellow)  AHG reagent
 Anti-D (clear)  22% bovine serum albumin
Procedures:  Check cells: IgG-coated group O red cells
1. Prepare 5% RBC suspension from EDTA WB  Incubator/ water bath: 37°C
2. Label 3 tubes: “A”, “B” and “D” Procedures:
3. Add 2 drops of each reagent into the same labeled tube  Prepare 1 labeled test tube
a. Anti-A for Tube A c. Anti-D for Tube D  Add 2 drops of serum and 1 drop RBC suspension to the test tube
b. Anti-B for Tube B
4. Add 2 drops of rbc suspension to each labeled tube (A, B, D) IMMEDIATE SPIN
5. Gently mix, allow the mixtures to stand at room temperature for 2 mins. 1. Mix gently, cover the tube with parafilm and let it stand in room temperature for 15 mins
6. Centrifuge at 3400 rpm at 10-15 sec (10-15 sec) 2. Centrifuge at 3400 rpm for 10-15 sec
7. Resuspend 3. Resuspend
8. Grade the reaction 4. Grade the result
Interpretation:
* IF NO AGGLUTINATION, PROCEED TO THE NEXT STEP *
Rh TYPING
 Rh-positive: agglutination THERMAL (37°C) INCUBATION PHASE
 Rh-negative: no agglutination 1. Add 2 drops of 22% bovine serum albumin to the previous nonreactive tube in immediate spin
- must be confirmed with weak D testing 2. Cover with parafilm and incubate at 37°C for 20 mins
3. Centrifuge at 3400 rpm for 10-15 sec
ABO BLOOD TYPING 4. Resuspend.
FORWARD REVERSE 5. Grade the result
Blood Group
Antigen (RBC) Antibody (Serum)
O No A or B antigen (-) Anti-A, Anti-B (+) * IF NO AGGLUTINATION, PROCEED TO THE NEXT STEP *
A A (+) Anti-B (+)
B B (+) Anti-A (+) AHG PHASE
AB A and B (+) No A or B antibodies (-) 1. Wash the tube 3 times with NSS (3/4 of the amount). Remove the supernatant.
2. Add 2 drops of AHG reagent. Cover with parafilm and mix
3. Centrifuge at 3400 rpm for 10-15 sec
ABO BLOOD TYPING
4. Grade the result.
REVERSE GROUPING (serum or back typing)
5. Add check cells to confirm its true negativity
Specimen:
Interpretation:
 Px serum (red-topped tube)
 At any phase, if there is an agglutination, test is (+) at that specific phase and no need to
Reagents:
proceed to another phase hence px serum is incompatible with donor’s RBC,
 A1 cells
 If there is no agglutination, at any phase, proceed to the following phase until AHG phase. Add
 B cells check cells if the result is still not reactive. Test is (-) hence px serum is compatible with
Procedures: donor’s RBC.
1. Label 2 test tubes: “A” and “B”  INTERPRETATION: Patient’s serum is in/compatible with donor’s RBC
2. Add 2drops of px serum into each labeled tube - COMPATIBLE: (-) in all tests
3. Add 2 drops of 5% rbc suspension into each labeled tube - INCOMPATIBLE: (+) in any phase
4. Gently mix, allow the mixtures to stand at room temperature for 2 mins
5. Centrifuge at 3400 rpm at 10-15 sec (10-15 sec)
6. Resuspend
7. Grade the reaction

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