CLS 3311Advanced Clinical Immunohematology

Antibody Identification

Additional Methods: Enzyme Treatment, Absorption, and Elution

Purpose of this Lecture

• Antibodies have a tendency to NOT read the textbooks and therefore, behave quite rudely at times.

• They also like to run in packs, i.e. multiple antibodies, and there are certain procedures that can help in those cases.

• Sometimes antibodies demonstrate only weak reactivity resulting in a very non descript pattern of reactivity. What can you do?

Weak, Non Descript Reactions

How can weak reactions be enhanced?1. Increase serum to cell ratio. Essentially adding

more antibody to the test system.

2. Reacts only at cold phase? Increase incubation time.

3. Enzyme treat panel cells. Rh, Kidd, I, and Lewis are ENHANCED by enzyme treatment.

4. AHG reactions only? Try another enhance- ment media such as PEG or another method like Gel.

Multiple Antibodies?

• Enzyme treatment of Panel red blood cells.

• Auto antibodies present in addition to allo antibodies? Auto Absorption. Antibody to high incidence antigen? Absorption with phenotypically similar red cells.

• Auto antibody with Positive DAT? Perform an Elution to identify auto antibody.

Enzyme Treatment of Red Blood Cells

• Enzymes such as Ficin, Papain, Bromelin, Neuraminidase, 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.

Enzyme Treated Red Blood Cells

• 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 treated 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. Antibody reactions to these antigens will be strengthened following enzyme treatment of red blood cells.

Enzyme Treatment of Panel Cells

• 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.

• Next, you need to know which antigens are destroyed by enzymes: Duffy, MNS(s), Xga. And which are enhanced by enzymes: Rh, Kidd, Lewis, I.

• The rule out process using the Enzyme treated panel MUST disregard those specificities that are destroyed by enzymes.

Does the patient serum reactivity weaken or strengthen after enzyme treatment of panel red cells

compared to non-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

Control 0 0 0 0 2+

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

Control 0 0 0 0 2+

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 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.

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

Control 0 0 0 0 2+

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 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

Control 0 0 0 0 2+

Enzyme Treated Panel

Enzyme Treated Panel Write Up

Untreated Panel Write Up1. 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.

Remember: Don’t look at Duffy, MNSs or Xga

Enzyme Panel Write Up

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

2. V, VS, Cw, Kpa, Jsa

3. Lea, Lua

4. Anti-C

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

• 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. Harmening

• 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.

Anti-e

Anti-e

Anti-e

Anti-S

Anti-e

Anti-S

Anti-e

Anti-e

Anti-e

e

Anti-S

ee

e

e

eee

e

e

ee

e ee

e

e

e

ee

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.

ee

e eee

e

e

eee

ee ee ee

Anti-e

Anti-eAnti-e

Anti-eAnti-e

Anti-eAnti-e

Anti-e

Anti-S

Anti-S

Anti-S

Anti-S

Antibody Adsorption: Application

1. Separating multiple antibodies present in a single serum.

2. Removing auto antibody activity to permit detection of underlying allo antibodies.

3. Removing unwanted antibody from a serum that contains an antibody suitable for reagent use.

4. Confirming the presence of specific antigens on red cells through their ability to remove antibody of corresponding specificity from previously characterized serum.

5. Confirming the specificity of an antibody by showing that it can be adsorbed only to red cells of a particular blood group phenotype.

Adsorption: Separating Multiple

Antibodies• Patient red cell phenotype is very helpful:

Phenotyping the patient can help in choosing a red cell phenotype that will give the most information.

• If None of the antibodies have been identified then a weak reacting cell can be used assuming that only one antibody may be reacting with it.

Adsorption: Separating Multiple

Antibodies• If One or More of the antibodies have been

identified, then red cells lacking those antigens are usually chosen so that only one antibody is removed.

• Large volumes of red cells are required so Reagent vials are insufficient. Donor blood or staff members end up being the most convenient.

Adsorption: Positive DAT

• Auto adsorption 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 determine degree of removal of auto antibody. May require second adsorption.

• Test panel with adsorbed serum to check for presence of allo antibody that was masked by the auto antibody.

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.

• Methods– Heat or Freeze thaw: used for ABO HDN– Acid or Organic solvent: warm reactive auto

and allo antibodies

Anti-e

Anti-e Anti-e

Anti-e

Anti-e

Anti-e

Anti-e eee

e e

eee

ee

ee

e ee

e

e

e

ee

Elution is the process of removal of antibody off of red cell membrane. Often, the red cell is destroyed in the process while

the antibody is left free in the eluate.

eee

ee

ee

ee

e

e ee ee ee

Anti-e

Anti-e

Anti-e

Anti-e

Anti-e

Anti-e

Anti-e Anti-e

Anti-e Anti-e

Antibody Elution: Application

• Investigation of Positive DAT– Hemolytic Disease of the Newborn (HDN),

Autoimmune hemolytic anemia, etc.

• Concentration and purification of antibody, the detection of weakly expressed antigens, and identification of multiple antibody specificities. Used in conjunction with adsorption technique.

• Preparation of antibody free red blood cells for use in phenotyping or autologous adsorption studies.

Adsorption-Elution Techniques

• Adsorb and Elute antibodies in a multiple antibody serum. Used to isolate individual antibodies.

• Serum should be Adsorbed until no more antibody reactivity is detected.

• Elution of adsorbed red blood cells is performed. A Panel is then tested with the eluate to determine the specificity of the eluted antibody.