Activity No.

________
CROSSMATCHING
I. INDIRECT ANTIGLOBULIN TEST

Principle and Application of the IAT:

A.) MAJOR

OBJECTIVES:
At the end of this activity, the student should be able to:

Principle and Application of the DAT:

Rationale

MATERIALS & EQUIPMENT

PROCEDURE:( USE the agglutination grading chart for the interpretation)

I. SPECIMEN COLLECTION
II. BLOOD COMPONENTS SEPARATION & RCS PREPARATION

III. CROSSMATCHING OF BLOOD

A. IMMEDIATE SPIN PHASE
1. Label tube with the patient’s name or identification number
2. Wearing gloves, place 2 drops of patient serum into the tube using a transfer pipet.
3. Add one drop of thoroughly mixed donor red cell suspension and mix gently.
4. Centrifuge for 15 seconds at 3400 rpm.
5. Examine the supernatant for hemolysis as tube is removed from the centrifuge.
6. Resuspend cells by gently agitating the tube while examining for agglutination with
a good light source. Grade the reaction.
1. Record result in the Immediate Spin Phase. If tube is positive, continue with the
next phase of testing.

B.BOVINE SERUM ALBUMIN ( BSA ) PHASE (may also use LISS or other enhancement media)
1. Add 2 drops of enhancement media or bovine serum albumin to the tube and mix
gently.
2. Incubate all tubes at 37oC for 30 minutes.
3. When incubation is complete, centrifuge the tubes for 15 seconds at 3400 rpm.

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 4. Examine the supernatant for hemolysis as tube is removed from the centrifuge.
5. Resuspend cells by gently agitating the tube while examining for agglutination with
a good light source. Grade the reaction.
1. Record result in the Bovine Serum Albumin Phase. If tube is positive, continue with
the next phase of testing.

C. ANTIHUMAN GLOBULIN ( AHG ) PHASE

1. Wash the serum/cell mixture from the previous phase with saline for three to four
times. (Centrifugation should be carried out at 3400 rpm for 1 minute.)
2. After the last washing, remove as much as saline from the tube by gently shaking
tube in an upside-down position. Blot dry the mouth of the tube with an absorbent
paper or gauze.
3. Add 2 drops of AHG reagent to the tube and mix gently.
4. Centrifuge tube for 15 seconds at 3400 rpm.
5. Examine the supernatant for hemolysis as tube is removed from the centrifuge.
6. Resuspend cells by gently agitating the tube while examining for agglutination with a
good light source. Grade the reaction.
7. Record result in the Antihuman Globulin Phase.
8. To the negative tube, add 2 drops of check cells.
9. Centrifuge for 15 seconds at 3400 rpm and examine supernatant for hemolysis.
10. Gently resuspend cells and observe for agglutination.
11. Record result in the check cell portion. If no agglutination is observed, the test
should be repeated.
12. Discard all biohazardous waste in a puncture-proof biohazard container.

Reporting of Results ( USE the agglutination grading chart for the interpretation)
Name of Patient IS BSA AHG Check cells Interpretation

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 Instructor’s Signature

B.) MINOR

OBJECTIVES:
At the end of this activity, the student should be able to:

MATERIALS & EQUIPMENT

PROCEDURE:( USE the agglutination grading chart for the interpretation)

I. SPECIMEN COLLECTION
II. BLOOD COMPONENTS SEPARATION & RCS PREPARATION
III. CROSSMATCHING OF BLOOD
IS
BSA
AHG

Reporting of Results ( USE the agglutination grading chart for the interpretation)
Name of Patient IS BSA AHG Check cells Interpretation

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 Activity No. ______
DIRECT ANTIGLOBULIN TEST
Principle and Application of the DAT:

Materials:

Procedure:

1. Label two 10 or 12 ( 75 mm glass test tubes. Test and control, respectively, and add 1 drop of a
3% v/v suspension oftest cells to each.
2. Wash the cells a minimum of three times with saline, and ensure that all saline is completely
decanted after the last wash.
3. To the tube labeled “test,” add 1 to 2 drops of AHG as recommended by the manufacturer and
mix.
4. To the control tube add 1 to 2 drops of 3% w/v bovine albumin in saline and mix.
5. Centrifuge both tubes at 1,500 RCF for 15 to 20 seconds.
6. Following centrifugation, completely resuspend the cell pellet by gently tipping and rolling the
tube. Read and score agglutination macroscopically with the aid of a background light source
and low-power magnification.
7. Incubate the tubes for another 5 minutes at room temperature and repeat steps 5 and 6.

Reporting of Results ( USE the agglutination grading chart for the interpretation)
Name of Patient LISS AHG

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