3. Add 1 mL of eluting solution and note the time.
4. Stopper the tube and mix by inversion for 90 seconds.
5. Remove the stopper and promptly centrifuge the tube
6. Transfer supernatant fluid to a clean test tube and add
5–6 drops of neutralizing solution; save red blood cells
for use in adsorption studies if needed.
7. Check pH; adjust it, if necessary, to pH 7.0 by adding
8. Centrifuge at 900–1000 g for 2–3 minutes to remove
precipitate that forms after neutralization. Harvest
the supernatant eluate and test it in parallel with
supernatant saline from final wash.
group antigens, except those of the Kell blood group
system. Expression of antigens in the Kell system is
markedly weakened after citric acid treatment.
2. Citric acid modified red blood cells may also be treated
with protease and used in autologous adsorption
Packed DAT positive red blood cells washed six times with
1. Glycine-HCl (0.1 M, pH 3.0), prepared by dissolving
3.75 g of glycine and 2.922 g of sodium chloride in
500 mL of distilled water. Adjust pH to 3.0 with 12N
2. Phosphate buffer (0.8 M, pH 8.2), prepared by
dissolving 109.6 g of Na2HPO4 and 3.8 g of KH2PO4 in
approximately 600 mL of distilled water. Adjust pH, if
necessary, with either 1N NaOH or 1N HCl. Dilute to a
final volume of 1 titer with distilled water. Store at 4°C.
4. Supernatant saline from final wash of red blood cells
1. Place the red blood cells in 13 × 100 mm test tube and
chill them in an ice bath for 5 minutes before adding
2. Add 1 mL of chilled saline and 2 mL of chilled glycineHCl to 1 mL of washed red blood cells.
3. Mix and incubate the tube in an ice bath for 1 minute.
4. Quickly centrifuge the tube at 900–1000 g for 2–3
5. Transfer the supernatant eluate into a clean test tube,
and add 0.1 mL of pH 8.2 phosphate buffer for each
6. Mix and centrifuge at 900–1000 g for 2–3 minutes.
7. Transfer the supernatant eluate into a clean test tube,
and add test in parallel with the supernatant saline
1. Keep glycine in ice bath during use, to maintain correct
2. Phosphate buffer will crystallize during storage at 4°C.
Redissolve it at 37°C before use.
3. Addition of phosphate buffer restores neutrality to
the acidic eluate. Unneutralized acidity may cause
hemolysis of the reagent red blood cells used in
testing the eluate. The addition of 22% bovine albumin
(one part to four parts of eluate) may reduce such
Packed DAT positive red blood cells washed six times with
1. Disodium EDTA (10% w/v): Na2EDTA2 H2O,10 g,
2. Glycine-HCl (0.1 M at pH 1.5): Glycine 3.754 g; NaCl
2.922 g; distilled water 500 mL; adjust to pH 1.5 with
3. TRIS base: Hydroxymethyl aminomethane, 12.1 g;
4. Supernatant saline from final wash of the red blood
1. Mix 4 mL of glycine-HCl and 1 mL of EDTA in 16 × 100
2. Immediately add 1 mL of washed red blood cells and
3. Incubate at room temperature for 1–2 minutes.
Blood Banking (Immunohematology) 351
4. Centrifuge the tube at 900–1000 g for 2–3 minutes.
5. Transfer the supernatant eluate into a clean test tube
and adjust to pH 7.5 with 1 M TRIS base.
6. Mix and centrifuge at 900–1000 g for 2–3 minutes.
7. Transfer the supernatant eluate into a clean test tube,
and test it in parallel with the supernatant saline from
1. Once the red blood cells have been rendered DATnegative, they may be tested for the presence of
blood group antigens, except those in the Kell system.
Treatment with glycine-HCl/EDTA denatures Kell
2. Red blood cells modified with glycine-HCl/EDTA
may be treated with protease and used in autologous
Packed DAT positive red blood cells washed six times with
1. Six percent bovine albumin, prepared by diluting 22%
or 30% bovine albumin with saline.
2. Supernatant saline from final wash of the red blood
1. Mix equal volumes of washed packed cells and 6%
bovine albumin in 13 × 100 mm test tube.
2. Place the tube at 56°C for 10 minutes. Agitate the tube
periodically during the incubation period.
3. Centrifuge the tube at 900–1000 g for 2–3 minutes,
preferably in a heated centrifuge.
4. Immediately transfer the supernatant eluate into a
clean test tube, and test in parallel with supernatant
Serum separated from freshly collected blood sample
1. Freshly collected normal serum, to use as a source of
2. 50% suspension of washed group O red blood cells that
1. Label three sets of three 10 × 75 mm test tubes as
follows: A1-A2-A3; B1-B2-B3; C1-C2-C3.
2. To tubes 1 and 2 of each set, add 10 volumes of the
3. To tubes 2 and 3 of each set, add 10 volumes of fresh
4. To all tubes, add one volume of 50% suspension of
washed P-positive red blood cells and mix well.
5. Place the three ‘A’ tubes in a bath of melting ice for 30
minutes, and then at 37°C for 1 hour.
6. Place the three ‘B’ tubes in a bath of melting ice, and
keep them in melting ice for 90 minutes.
7. Place the three ‘C’ tubes at 37°C, and keep them at 37°C
8. Centrifuge all tubes, and examine the supernatant fluid
The Donath-Landsteiner test is considered positive when
the patient serum, with or without added complement,
causes hemolysis in the tubes that were incubated first in
melting ice and then at 37°C (i.e. tubes A1 and A2), and there
is no hemolysis in any of the tubes maintained throughout
at 37°C or in melting ice. The A3, B3 and C3 tubes serve as a
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