5. Donor cells/reagent red blood cells.
1. To properly labeled test tubes add two drops of serum.
2. Add an equivalent volume of 22 or 30% bovine albumin
(unless manufacturer’s directions state otherwise).
3. Add one drop of 2 to 5% saline suspended reagent or
donor red blood cells to each tube and mix.
4. Incubate at 37°C for 15 to 30 minutes.
5. Centrifuge for 15 to 20 seconds at 900 to 1000 g.
Observe for hemolysis and/or agglutination. Grade
Blood Banking (Immunohematology) 345
6. Wash the cells three or four times with saline and
completely decant after final wash.
7. Add AHG to cell button according to the manufacturer’s
8. Centrifuge and observe for reaction. Grade and record
9. Confirm the validity of negative tests by adding
LISS (Low Ionic Strength Solution) Phase Indirect Antihuman Globulin Test
Serum or plasma may be used. Preferably, freshly collected
3. Polyspecific AHG or monospecific anti-human IgG
5. Donor cells/reagent red blood cells.
1. Wash reagent or donor red blood cells three times in
normal saline and completely decant saline after last
2. Resuspend the cells to a 2–5% suspension in LISS.
3. To properly labeled test tube, add two drops of serum.
4. Add two drops of LISS suspended red blood cell
suspension and incubate according to manufacturer’s
direction. Typically, this is 10 to 15 minutes at 37°C.
5. Centrifuge according to manufacturer’s direction.
Typically, this is 15 to 30 seconds at 900 to 1000 g and
observe for hemolysis and agglutination by gently
resuspending the cell button. Grade and record results.
6. Wash the cells three or four times with saline and
completely decant after final wash.
7. Add AHG to cell button according to the manufacturer’s
8. Centrifuge for 15 to 20 seconds at 900 to 1000 g and
observe for reaction. Grade and record the results.
9. Confirm the validity of negative tests by adding
PEG-enhanced Indirect Anti-human Globulin Test
Serum or plasma may be used. Preferably, freshly collected
3. Polyspecific AHG or monospecific anti-human IgG
5. Donor cells/reagent red blood cells.
1. For each sample to be tested, mix 2 drops of test serum,
4 drops of 20% PEG in PBS, and 1 drop of 2–5% red
2. Incubate according to manufacturer’s directions.
Typically, this is 15 minutes at 37°C.
4. Wash the cells four times with saline and completely
5. Add AHG to cell button according to the manufacturer’s instruction. Mix well.
6. Centrifuge for 15 to 20 seconds at 900 to 1000 g and
observe for reaction. Grade and record the results.
7. Confirm the validity of negative tests by adding
LIM (Low Ionic Medium Polybrene) Indirect Antihuman Globulin Test
Serum or plasma may be used. Preferably, freshly collected
2. Low ionic medium (LIM): To a 500 mL volumetric
flask, add 25 g of dextrose and 1 g of Na2 EDTA 2H2O.
Fill flask to 500 mL mark with distilled water.
3. Polybrene (Commercially available).
4. Resuspending medium: 0.2 M trisodium citrate, 5%
dextrose; working solution made by mixing 60 mL of
0.2 M trisodium citrate with 40 mL of 5% dextrose.
5. Washing solution (for anti-human globulin testing):
6. Polyspecific AHG or monospecific anti-human IgG
8. Donor cells/reagent red blood cells.
1. Prepare 1% suspension of donor or reagent red blood
cells in the serum used for testing.
2. Add 1.0 mL of LIM solution. Mix and incubate for
3. Add 0.1 mL of 0.05% Polybrene to each tube and mix.
4. Centrifuge according to manufacturer’s directions.
Typically, this is 10 seconds at 900 to 1000 g and
decants the supernatant fluid. Do not resuspend cell
5. Add 0.1 mL of resuspending solution. Shake tube
gently and observe for persistent agglutination.
and a known negative control macroscopically. Do
6. If desired, the anti-human globulin test may be
a. Add 0.05 mL of resuspending solution to each
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