Advanced Concepts in

Immunohematology
Antibody Identification
Additional Methods: Enzyme Treatment, Adsorption, Elution
and Other Techniques
 How can weak reactions be enhanced?
Antibody
Identification
Additional Increase serum to cell ratio. Essentially adding
more antibody to the test system.
Methods:
Enzyme Reacts only at cold phase? Increase incubation
Treatment, time.
Adsorption,
Elution and Enzyme treat panel cells. Rh, Kidd, I, and Lewis
are ENHANCED by enzyme treatment.
Other
Techniques AHG reactions only? Try another enhance- ment
media such as PEG or another method like Gel.
 Enzyme treatment of Panel red blood cells.

Multiple Auto antibodies present in addition to allo

antibodies? Autoabsorption. Antibody to

Antibodies?
high incidence antigen? Absorption with
phenotypically similar red cells.

Auto antibody with Positive DAT? Perform

an Elution to identify autoantibody.
Enzyme Treatment of
Red Blood Cells
Enzymes such as Ficin, Papain, Bromelin, Trypsin, etc. are routinely used
in the blood bank to assist in the antibody identification process.

Enzymes are used to help differentiate antibody specificities in patients

with multiple antibodies.

Not all enzymes cleave the same antigens. A knowledge of each Blood
Group and the how enzymes affect them is necessary.
 • Enzymes Destroy (cleave off) some antigens
from the red cell membrane including Duffy
(Fya, Fyb), Xga, M, N, S and s. Variable
reactions are seen with s antigens. Antibodies
to these antigens will NOT react with enzyme

Enzyme Treated
treated red blood cells.

Red Blood Cells • Enzymes Enhance the reactivity of some

blood groups by giving their antigens greater
exposure to their corresponding antibody
including Kidd, Rh, Lewis and Ii, P1. Antibody
reactions to these antigens will be
strengthened following enzyme treatment of
red blood cells.
 One-stage

• Enzyme is added directly to

the serum/cell mixture
Enzyme
techniques Two-stage

• Panel cells are pre-treated

with enzyme, incubated and
washed
• Patient serum is added to
panel cells and tested
 First, it is necessary to have both an untreated
and treated panel of the same lot #. The only
way to see if an antibodies reactivity is altered is
to compare the enzyme treated panel reactions
with an untreated panel.

Enzyme Next, you need to know which antigens are

Treatment destroyed by enzymes: Duffy, MNS(s), Xga. And
which are enhanced by enzymes: Rh, Kidd,

of Panel Cells
Lewis, I.

The rule out process using the Enzyme treated

panel MUST disregard those specificities that are
destroyed by enzymes.
Enzyme treated panel cells.
Rh MNSs P Lewis Lutheran Kell Duffy Kidd

D C E c e f V VS CW M N S s P1 Lea Leb Lua Lub K k Kpa Jsa Fya Fyb Jka Jkb Xga IS RT 37 AHG CC

1 0 + 0 + + + 0 0 0 + + + 0 0 + 0 + + 0 + 0 0 + + + + + 0 0 0 1+

2 + + 0 0 + 0 0 0 + 0 + 0 + + + 0 0 + 0 + 0 0 + 0 + + + 0 0 0 2+

3 + + 0 0 + 0 0 0 0 + + 0 + + 0 + 0 + + + 0 0 0 + 0 + + 0 0 0 1+

4 + 0 + + O 0 0 / 0 + 0 0 + + 0 0 0 + 0 + 0 0 + 0 + 0 + 0 0 0 2+

5 0 0 + + + + 0 / 0 + 0 + + + 0 + 0 + 0 + 0 0 + + + + + 0 0 0 1+

6 0 0 0 + + + + + 0 0 + 0 + + 0 + 0 + 0 + 0 0 + 0 + 0 0 0 0 0 2+

7 0 0 0 + + + 0 0 0 + 0 0 + W 0 + 0 + + 0 0 0 0 + + + 0 0 0 0 1+

8 0 0 0 + + + 0 0 0 + + 0 + + 0 0 0 + 0 + 0 + + + + 0 + 0 0 0 1+w

9 0 0 0 + + + 0 / 0 + + + + 0 0 + 0 + 0 W + 0 + 0 + 0 + 0 0 0 2+

10 0 0 0 + + + 0 0 0 0 + 0 + + 0 + 0 + 0 + 0 0 + + 0 + + 0 0 0 1+

11 + + 0 0 + / 0 / 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + + 0 0 0 1+
Auto
0 0 0 0 2+
Control

Does the patient serum reactivity weaken or strengthen after

enzyme treatment of panel red cells compared to non-enzyme
treated panel cells?
 Enzyme treated panel cells.
Rh MNSs P Lewis Lutheran Kell Duffy Kidd

D C E c e f V VS CW M N S s P1 Lea Leb Lua Lub K k Kpa Jsa Fya Fyb Jka Jkb Xga IS RT 37 AHG CC

1 0 + 0 + + + 0 0 0 + + + 0 0 + 0 + + 0 + 0 0 + + + + + 0 0 0 0 2+

2 + + 0 0 + 0 0 0 + 0 + 0 + + + 0 0 + 0 + 0 0 + 0 + + + 0 0 0 0 2+

3 + + 0 0 + 0 0 0 0 + + 0 + + 0 + 0 + + + 0 0 0 + 0 + + 0 0 0 1+

4 + 0 + + O 0 0 / 0 + 0 0 + + 0 0 0 + 0 + 0 0 0 0 + 0 + 0 0 0 0 2+

5 0 0 + + + + 0 / 0 + 0 + + + 0 + 0 + 0 + 0 0 + + + + + 0 0 0 0 2+

6 0 0 0 + + + + + 0 0 + 0 + + 0 + 0 + 0 + 0 0 + 0 + 0 0 0 0 0 0 2+

7 0 0 0 + + + 0 0 0 + 0 0 + W 0 + 0 + + 0 0 0 0 + + + 0 0 0 0 1+

8 0 0 0 + + + 0 0 0 + + 0 + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 0 0 0 2+

9 0 0 0 + + + 0 / 0 + + + + 0 0 + 0 + 0 W + 0 + 0 + 0 + 0 0 0 0 2+

10 0 0 0 + + + 0 0 0 0 + 0 + + 0 + 0 + 0 + 0 0 + + 0 + + 0 0 0 0 2+

11 + + 0 0 + / 0 / 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + + 0 0 0 0 2+
Auto
0 0 0 0 2+
Control

Blood groups highlighted with light blue are those that are
destroyed by enzymes and those highlighted with purple are
enhanced by enzyme treatment of red blood cells.
Enzyme Treated
Panel
• The next two Panel’s represent the process of
enzyme treating red blood cells.
• First Panel: Test patient serum with untreated
panel.
• Second Panel: Test serum with Enzyme treated
panel.
• Print each panel and go through the rule out
process. Write up each panel.
• A write up and explanation will follow.
 Untreated Panel
Rh MNSs Lewis Lutheran Kell Duffy Kidd

D C E c e f V VS Cw M N S s P1 Lea Leb Lua Lub K k Kpa Jsa Fya Fyb Jka Jkb Xga IS RT 37 AHG CC

1 0 + 0 + + + 0 0 0 + + + 0 0 + 0 + + 0 + 0 0 + + + + + 0 0 0 1+

2 + + 0 0 + 0 0 0 + 0 + 0 + + + 0 0 + 0 + 0 0 + 0 + + + 0 0 0 3+

3 + + 0 0 + 0 0 0 0 + + 0 + + 0 + 0 + + + 0 0 0 + 0 + + 0 0 0 1+

4 + 0 + + O 0 0 / 0 + 0 0 + + 0 0 0 + 0 + 0 0 + 0 + 0 + 0 0 0 2+

5 0 0 + + + + 0 / 0 + 0 + + + 0 + 0 + 0 + 0 0 + + + + + 0 0 0 W+

6 0 0 0 + + + + + 0 0 + 0 + + 0 + 0 + 0 + 0 0 + 0 + 0 0 0 0 0 2+

7 0 0 0 + + + 0 0 0 + 0 0 + W 0 + 0 + + 0 0 0 0 + + + 0 0 0 0 0 2+

8 0 0 0 + + + 0 0 0 + + 0 + + 0 0 0 + 0 + 0 + 0 + + 0 + 0 0 0 0 2+

9 0 0 0 + + + 0 / 0 + + + + 0 0 + 0 + 0 W + 0 + 0 + 0 + 0 0 0 2+

10 0 0 0 + + + 0 0 0 0 + 0 + + 0 + 0 + 0 + 0 0 + + 0 + + 0 0 0 W+

11 + + 0 0 + / 0 / 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + + 0 0 0 1+
Auto
0 0 0 0 2+
Control
 Enzyme Treated
Panel
Rh MNSs Lewis Lutheran Kell Duffy Kidd

D C E c e f V VS Cw M N S s P1 Lea Leb Lua Lub K k Kpa Jsa Fya Fyb Jka Jkb Xga IS RT 37 AHG CC

1 0 + 0 + + + 0 0 0 + + + 0 0 + 0 + + 0 + 0 0 + + + + + 0 0 0 1+

2 + + 0 0 + 0 0 0 + 0 + 0 + + + 0 0 + 0 + 0 0 + 0 + + + 0 0 0 1+

3 + + 0 0 + 0 0 0 0 + + 0 + + 0 + 0 + + + 0 0 0 + 0 + + 0 0 0 1+

4 + 0 + + O 0 0 / 0 + 0 0 + + 0 0 0 + 0 + 0 0 0 0 + 0 + 0 0 0 0 2+

5 0 0 + + + + 0 / 0 + 0 + + + 0 + 0 + 0 + 0 0 + + + + + 0 0 0 0 2+

6 0 0 0 + + + + + 0 0 + 0 + + 0 + 0 + 0 + 0 0 + 0 + 0 0 0 0 0 0 2+

7 0 0 0 + + + 0 0 0 + 0 0 + W 0 + 0 + + 0 0 0 0 + + + 0 0 0 0 0 2+

8 0 0 0 + + + 0 0 0 + + 0 + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 0 0 0 2+

9 0 0 0 + + + 0 / 0 + + + + 0 0 + 0 + 0 W + 0 + 0 + 0 + 0 0 0 0 2+

10 0 0 0 + + + 0 0 0 0 + 0 + + 0 + 0 + 0 + 0 0 + + 0 + + 0 0 0 0 2+

11 + + 0 0 + / 0 / 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + + 0 0 0 1+
Auto
0 0 0 0 2+
Control
Enzyme Treated Panel Write Up
Untreated Panel Write Up

1. D, C, E, V, VS, Cw, M, N, S, Lea, Leb, Lua, K, k, Kpa, Jsa, Fya, Jka, Jkb, Xga

2. V, VS, Cw, Kpa, Jsa

3. M, N, Lea, Leb, Lua

4. ?????

5. ?????

At this point it is very difficult to see a pattern that stands out. Do the Enzyme treated panel write up and see if that helps.
 Enzyme treated Panel
Rh MNSs Lewis Lutheran Kell Duffy Kidd

D C E c e f V VS Cw M N S s P1 Lea Leb Lua Lub K k Kpa Jsa Fya Fyb Jka Jkb Xga IS RT 37 AHG CC

1 0 + 0 + + + 0 0 0 + + + 0 0 + 0 + + 0 + 0 0 + + + + + 0 0 0 1+

2 + + 0 0 + 0 0 0 + 0 + 0 + + + 0 0 + 0 + 0 0 + 0 + + + 0 0 0 1+

3 + + 0 0 + 0 0 0 0 + + 0 + + 0 + 0 + + + 0 0 0 + 0 + + 0 0 0 1+

4 + 0 + + O 0 0 / 0 + 0 0 + + 0 0 0 + 0 + 0 0 0 0 + 0 + 0 0 0 0 2+

5 0 0 + + + + 0 / 0 + 0 + + + 0 + 0 + 0 + 0 0 + + + + + 0 0 0 0 2+

6 0 0 0 + + + + + 0 0 + 0 + + 0 + 0 + 0 + 0 0 + 0 + 0 0 0 0 0 0 2+

7 0 0 0 + + + 0 0 0 + 0 0 + W 0 + 0 + + 0 0 0 0 + + + 0 0 0 0 0 2+

8 0 0 0 + + + 0 0 0 + + 0 + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 0 0 0 2+

9 0 0 0 + + + 0 / 0 + + + + 0 0 + 0 + 0 W + 0 + 0 + 0 + 0 0 0 0 2+

10 0 0 0 + + + 0 0 0 0 + 0 + + 0 + 0 + 0 + 0 0 + + 0 + + 0 0 0 0 2+

11 + + 0 0 + / 0 / 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + + 0 0 0 1+
Auto
0 0 0 0 2+
Control
 Enzyme Panel Write Up

1. D, C, V, VS, Cw, Lea, Lua, K, Kpa, Jsa,

Jkb
Remember: 2. V, VS, Cw, Kpa, Jsa
Don’t look at
Duffy, MNSs 3. Lea, Lua
or Xga
4. Anti-C

5. Big C negative, D+, K+, Jkb+ so far…

 Untreated Panel
Rh MNSs Lewis Lutheran Kell Duffy Kidd

D C E c e f V VS Cw M N S s P1 Lea Leb Lua Lub K k Kpa Jsa Fya Fyb Jka Jkb Xga IS RT 37 AHG CC

1 0 + 0 + + + 0 0 0 + + + 0 0 + 0 + + 0 + 0 0 + + + + + 0 0 0 1+

2 + + 0 0 + 0 0 0 + 0 + 0 + + + 0 0 + 0 + 0 0 + 0 + + + 0 0 0 3+

3 + + 0 0 + 0 0 0 0 + + 0 + + 0 + 0 + + + 0 0 0 + 0 + + 0 0 0 1+

4 + 0 + + O 0 0 / 0 + 0 0 + + 0 0 0 + 0 + 0 0 + 0 + 0 + 0 0 0 2+

5 0 0 + + + + 0 / 0 + 0 + + + 0 + 0 + 0 + 0 0 + + + + + 0 0 0 W+

6 0 0 0 + + + + + 0 0 + 0 + + 0 + 0 + 0 + 0 0 + 0 + 0 0 0 0 0 2+

7 0 0 0 + + + 0 0 0 + 0 0 + W 0 + 0 + + 0 0 0 0 + + + 0 0 0 0 0 2+

8 0 0 0 + + + 0 0 0 + + 0 + + 0 0 0 + 0 + 0 + 0 + + 0 + 0 0 0 0 2+

9 0 0 0 + + + 0 / 0 + + + + 0 0 + 0 + 0 W + 0 + 0 + 0 + 0 0 0 2+

10 0 0 0 + + + 0 0 0 0 + 0 + + 0 + 0 + 0 + 0 0 + + 0 + + 0 0 0 W+

11 + + 0 0 + / 0 / 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + + 0 0 0 1+
Auto
0 0 0 0 2+
Control
 Untreated Panel
Rh MNSs Lewis Lutheran Kell Duffy Kidd

D C E c e f V VS Cw M N S s P1 Lea Leb Lua Lub K k Kpa Jsa Fya Fyb Jka Jkb Xg a IS RT 37 AHG CC

1 0 + 0 + + + 0 0 0 + + + 0 0 + 0 + + 0 + 0 0 + + + + + 0 0 0 1+

2 + + 0 0 + 0 0 0 + 0 + 0 + + + 0 0 + 0 + 0 0 + 0 + + + 0 0 0 3+

3 + + 0 0 + 0 0 0 0 + + 0 + + 0 + 0 + + + 0 0 0 + 0 + + 0 0 0 1+

4 + 0 + + O 0 0 / 0 + 0 0 + + 0 0 0 + 0 + 0 0 + 0 + 0 + 0 0 0 2+

5 0 0 + + + + 0 / 0 + 0 + + + 0 + 0 + 0 + 0 0 + + + + + 0 0 0 W+

6 0 0 0 + + + + + 0 0 + 0 + + 0 + 0 + 0 + 0 0 + 0 + 0 0 0 0 0 2+

7 0 0 0 + + + 0 0 0 + 0 0 + W 0 + 0 + + 0 0 0 0 + + + 0 0 0 0 0 2+

8 0 0 0 + + + 0 0 0 + + 0 + + 0 0 0 + 0 + 0 + 0 + + 0 + 0 0 0 0 2+

9 0 0 0 + + + 0 / 0 + + + + 0 0 + 0 + 0 W + 0 + 0 + 0 + 0 0 0 2+

10 0 0 0 + + + 0 0 0 0 + 0 + + 0 + 0 + 0 + 0 0 + + 0 + + 0 0 0 W+

11 + + 0 0 + / 0 / 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + + 0 0 0 1+
Auto
0 0 0 0 2+
Control
 D, C, E, V, VS, Cw, M, N, S, Lea, Leb, Lua, K, k,
Kpa, Jsa, a
Fy , Jka, Jkb, Xga
Rh MNSs Lewis Lutheran Kell Duffy Kidd

D C E c e f V VS Cw M N S s P1 Lea Leb Lua Lub K k Kpa Jsa Fya Fyb Jka Jkb Xg a IS RT 37 AHG CC

1 0 + 0 + + + 0 0 0 + + + 0 0 + 0 + + 0 + 0 0 + + + + + 0 0 0 1+

2 + + 0 0 + 0 0 0 + 0 + 0 + + + 0 0 + 0 + 0 0 + 0 + + + 0 0 0 3+

3 + + 0 0 + 0 0 0 0 + + 0 + + 0 + 0 + + + 0 0 0 + 0 + + 0 0 0 1+

4 + 0 + + O 0 0 / 0 + 0 0 + + 0 0 0 + 0 + 0 0 + 0 + 0 + 0 0 0 2+

5 0 0 + + + + 0 / 0 + 0 + + + 0 + 0 + 0 + 0 0 + + + + + 0 0 0 W+

6 0 0 0 + + + + + 0 0 + 0 + + 0 + 0 + 0 + 0 0 + 0 + 0 0 0 0 0 2+

7 0 0 0 + + + 0 0 0 + 0 0 + W 0 + 0 + + 0 0 0 0 + + + 0 0 0 0 0 2+

8 0 0 0 + + + 0 0 0 + + 0 + + 0 0 0 + 0 + 0 + 0 + + 0 + 0 0 0 0 2+

9 0 0 0 + + + 0 / 0 + + + + 0 0 + 0 + 0 W + 0 + 0 + 0 + 0 0 0 2+

10 0 0 0 + + + 0 0 0 0 + 0 + + 0 + 0 + 0 + 0 0 + + 0 + + 0 0 0 W+

11 + + 0 0 + / 0 / 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + + 0 0 0 1+
Auto
0 0 0 0 2+
Control
 Untreated Panel
Rh MNSs Lewis Lutheran Kell Duffy Kidd

D C E c e f V VS Cw M N S s P1 Lea Leb Lua Lub K k Kpa Jsa Fya Fyb Jka Jkb Xga IS RT 37 AHG CC

1 0 + 0 + + + 0 0 0 + + + 0 0 + 0 + + 0 + 0 0 + + + + + 0 0 0 1+

2 + + 0 0 + 0 0 0 + 0 + 0 + + + 0 0 + 0 + 0 0 + 0 + + + 0 0 0 3+

3 + + 0 0 + 0 0 0 0 + + 0 + + 0 + 0 + + + 0 0 0 + 0 + + 0 0 0 1+

4 + 0 + + O 0 0 / 0 + 0 0 + + 0 0 0 + 0 + 0 0 + 0 + 0 + 0 0 0 2+

5 0 0 + + + + 0 / 0 + 0 + + + 0 + 0 + 0 + 0 0 + + + + + 0 0 0 W+

6 0 0 0 + + + + + 0 0 + 0 + + 0 + 0 + 0 + 0 0 + 0 + 0 0 0 0 0 2+

7 0 0 0 + + + 0 0 0 + 0 0 + W 0 + 0 + + 0 0 0 0 + + + 0 0 0 0 0 2+

8 0 0 0 + + + 0 0 0 + + 0 + + 0 0 0 + 0 + 0 + 0 + + 0 + 0 0 0 0 2+

9 0 0 0 + + + 0 / 0 + + + + 0 0 + 0 + 0 W + 0 + 0 + 0 + 0 0 0 2+

10 0 0 0 + + + 0 0 0 0 + 0 + + 0 + 0 + 0 + 0 0 + + 0 + + 0 0 0 W+

11 + + 0 0 + / 0 / 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + + 0 0 0 1+
Auto
0 0 0 0 2+
Control
Now, return to the Untreated Panel knowing that anti-C is present.

First look at the blood groups that are destroyed by enzymes: M, N, S, s, Fya, Fyb
and Xga.
• M & N are cold reacting, so they are not causing the additional reactions.
• Little s and Fyb have already been ruled out.

That leaves S, Fya and Xga. Now it is a matter of matching the reaction pattern with
the remaining specificities.

The most likely antibodies are anti-C and anti-Fya

The Selected Cell Panel must be negative for C and Fya antigens and positive for D,
K, S, Xga, & Jkb. Look at both panels to determine which need to be ruled out.
Antibody Adsorption

Definition: The removal of antibody from

serum by combining a serum sample with
appropriate RBCs under optimal conditions.

Antibody can be removed from a serum by

adsorption to red cells carrying the
corresponding antigen.

Serum and cells are mixed. After the antibody

attaches to the membrane bound antigens the
serum and cells are separated, the antibody
remains attached to the RBCs.
Mix serum with antibody with red cells with corresponding antigen.
Incubate at appropriate temperature. Anti-e antibodies have been
adsorbed out of the serum and onto the RBCs.
 Antibody Adsorption: Application

Separating Separating multiple antibodies present in a single serum.

Removing Removing autoantibody activity to permit detection of underlying alloantibodies.

Removing unwanted antibody from a serum that contains an antibody suitable

Removing
for reagent use.
Confirming the presence of specific antigens on red cells through their ability to
Confirming remove antibody of corresponding specificity from previously characterized
serum.
Confirming the specificity of an antibody by showing that it can be adsorbed only
Confirming
to red cells of a particular blood group phenotype.
Adsorption

• Two types:
–Autoadsorption
• No recent transfusion
• Autoantibodies are removed using patient RBCs, so alloantibodies can be
identified

–Allogeneic (Differential) adsorption

• If recently transfused
• Uses other cells with the patient’s serum
2
Tubes
Adsorption: Positive DAT

Autoadsorption is simply
mixing patient serum with
patient red cells to remove
the auto antibody remaining
in the serum under optimal
conditions. If it is a warm
auto antibody then adsorb
at warm temps, if it is a cold
auto antibody then adsorb
at cold temps.
Retest serum to Test panel with
determine degree of adsorbed serum to
removal of auto check for presence of
antibody. May require alloantibody that was
second adsorption. masked by the auto
antibody.
CASE 1

If the technologist wants

to separate anti-K and
anti-c so that anti-c will
ANS: K+c-
remain in the
supernatant, what type
of cells must be used?
 Anti-E and Anti-Fyb are two
possible antibodies implicated
in a case of HDFN. If the
technologist wants to confirm if
the reactivity is due to Anti-Fyb
CASE 2 through adsorption, what type
of red cells must be used?

ANS: Fy(b+), E-
 • Elution techniques “free”
Elution (whenever antibodies from the
DAT is positive) sensitized red cells so that
the antibodies can be
identified
 Antibody Elution

Definition: Elution is the removal of antibodies bound to

red cell membrane. The objective is to recover the
antibody in a usable form. In other words, we want it still
functional so it can be identified.
Elution is the process of removal of
antibody off red cell membrane. Often,
the red cell is destroyed in the process
while the antibody is left free in the
eluate.
Elution

1 2 3 4
The eluate is a term Testing the eluate is The red cells can also When tested with panel
used for the removed useful in investigations be used after elution for cells, the eluate usually
antibodies of positive DATs RBC phenotyping if remains reactive with all
• HDN needed cells if a warm
• Transfusion reactions autoantibody is present
• Autoimmune disease
 Acid elutions (glycine acid)

• Most common
• Lowers pH (3.0), causing antibody to
dissociate (e.g. citric acid and digitonin)

Organic solvents (ether, chloroform)

Elution • Dissolve bilipid layer of RBC
Methods
Heat (conformational change)

• 45°C: partial
ABO • 56°C: total
antibodies

Freeze-Thaw (lyses cells)

 Investigation of Positive DAT
• Hemolytic Disease of the Newborn (HDN), Autoimmune
hemolytic anemia, etc.

Antibody Concentration and purification of antibody, the

detection of weakly expressed antigens, and
Elution: identification of multiple antibody specificities.
Used in conjunction with adsorption technique.
Application
Preparation of antibody free red blood cells for
use in phenotyping or autologous adsorption
studies.
 Adsorb and Elute antibodies in a
multiple antibody serum. Used to isolate
individual antibodies.

Adsorption- Serum should be Adsorbed until no

more antibody reactivity is detected.
Elution
Techniques
Elution of adsorbed red blood cells is
performed. A Panel is then tested with
the eluate to determine the specificity of
the eluted antibody.
Many of elution tests can damage the antigens on
the RBC

Chloroquine diphosphate (CDP) and glycine acid

EDTA reagents can dissociate IgG from the RBC
without damaging the antigens
• Very useful if the RBC needs to be antigen typed
• NOTE: glycine/EDTA destroys KELL antigens
 Quinilone derivative often used as
an antimalarial

Chloroquine May not remove autoantibody

completely from DAT positive cells
diphosphate

Partial removal may be enough to

antigen type the cells or to be used
for autoadsorption of warm
autoantibodies
Neutralization
Some antibodies may be neutralized as a way of confirmation

Commercial “substances” bind to the antibodies in the patient serum, causing them to show no
reaction when tested with the corresponding antigen (in panel)

Manufacturer’s directions should be followed and a dilutional control should always be used

• The control contains saline and serum (no substance) and should remain positive
• A control shows that a loss of reactivity is due to the neutralization and not to the dilution of the antibody
strength when the substance is added

Common substances
• P1 substance (sometimes derived from hydatid cyst fluid or pigeon egg white)
• Lea and Leb substance (soluble antigen found in plasma and saliva)
• I substance can be found in breast milk
• Sda substance derived from human or guinea pig urine
• **you should be aware that many of these substances neutralize COLD antibodies; Cold antibodies can
sometimes mask more clinically significant antibodies (IgG), an important reason to use neutralization
techniques
 Sulfhydryl Reagents

Cleave the disulfide bonds of IgM molecules and

help differentiate between IgM and IgG antibodies

Good to use when you have both IgG and IgM

antibodies (warm/cold)
• Dithiothreitol (DTT) is a thiol and will denature Kell antigens
• 2-mercaptoethanol (2-ME)
ZZAP

A combination of proteolytic enzymes and DTT

Denatures Kell, M, N, S, Duffy and other less frequent blood group antigens

Does not denature the Kx antigen

“frees” autoantibody off patient’s cell, so that

Good for adsorption techniques autoantibody can then be adsorbed onto another RBC
Warm alloantibody
Cold alloantibody
Warm Autoantibody
Multiple Antibody
Rouleaux
Cold
Autoantibody/Antibody to
high incidence antigen
Multiple
antibody/Antibody to
high incidence antigen