DETECTION AND IDENTIFICATION OF ANTIBODIES

READ THE QUESTIONS CAREFULLY.

Detection of antibodies against RBC antigens

It is one of the principal tools for investigating potential hemolytic transfusion reactions and immune hemolytic anemias.

The focus of antibody detection methods is on “irregular” or “unexpected” antibodies, as opposed to the “expected” antibodies of the ABO system

Modified True or False.

The focus of antibody detection methods is on “irregular” or “unexpected” antibodies, similar to the antibodies of the ABO system.

Immune alloantibodies

What are the unexpected antibodies or primary importance?

IAT in test tube

The traditional testing method used to detect clinically significant antibodies.

True

Modified True or False.

In IAT method, RBC reagents (suspended at a concentration between 2% and 5% in a preservative diluent), enhancement reagent (if desired), and AHG reagent are used to sensitize reagent RBCs with the patient’s antibodies, followed by formation of visible RBC agglutinates.

Immediate spin phase

It is done to detect antibodies reacting at room temperature. It is done through centrifugation.

37 C incubation phase

In this phase, the IgG antibodies, if present in the serum, will sensitize any reagent RBCs that possess the target antigen.

False.

Tubes only need to be observed for hemolysis when fresh serum and reagent RBCs that do not contain EDTA are used because complement is necessary for hemolysis to occur.

Modified True or False.

Tubes only need to be observed for hemolysis when fresh serum and reagent RBCs that contain EDTA are used because complement is necessary for hemolysis to occur.

False.

All negative tests must have Coombs’ control cells (also known as check cells) added to confirm the validity of the negative result.

Modified True or False.

All positive tests must have Coombs’ control cells (also known as check cells) added to confirm the validity of the result.

• Flexible

• Availability of required testing equipment

• Relative low cost

Advantages of using Tube method

• Instability of reactions

• Subjective nature of grading

• Amount of hands-on time

• Problems can arise from washing phase

Disadvantages of using Tube method

True

Modified True or False.

The RBC reagents used in antibody screen testing come from group O individuals who have been typed for the most common and the most significant RBC antigens.

False.

Group O cells are used so that anti-A and anti-B will not interfere in the detection of antibodies to other blood group systems

Modified True or False.

Group O cells are used so that anti-A and anti-B can also react during the detection of antibodies to other blood group systems.

C

It contains more than one RBC population, hence it should be carefully observed for mixed-field agglutination

A pooled RBC screening reagent cannot be used for patient antibody screening, only for donor screening.

It contains more than one RBC population, but its specificity does not allow mixed field agglutination to form.

---

A. Both statements are correct

B. Both statements are incorrect

C. First statement is correct, second statement is incorrect

D. First statement is incorrect, second statement is correct

Homozygous

An expression present in individuals with only one allele at a given genetic locus. It has a double dose of the antigen.

Heterozygous

An expression present in individuals with two different alleles at a given genetic locus. The alleles share the available antigen,

Enhancement reagents / Potentiators

It is used to increase sensitivity and shorten incubation time. It is added prior to 37 C incubation phase.

• 22% albumin

• LISS

• PEG

What are the enhancement reagents used in IAT?

AHG Reagents

It is added to the test system as it allows for agglutination of incomplete antibodies.

Polyvalent or Broad Spectrum Coomb’s Serum

Polyspecific AHG reagent contains antibodies to both IgG and complement components. It is also called?

True

Modified True or False.

Polyspecific AHG reagent is not often used when performing antibody detection testing since the presence of anticomplement in the AHG reagent may lead to the detection of clinically insignificant anti- bodies.

Coomb’s Control

These are Rh-positive RBCs that have been coated with anti-D.

True

Modified True or False.

In a negative antiglobulin test, these antibody-coated cells (Coomb’s control) will react with the anti-IgG in the AHG reagent that remains free in the test tube, resulting in visible agglutination.

• Gel technology

• Solid-Phase technology

Other than AHG / Coomb’s Check cells, what other methods are routinely performed for antibody screening?

False.

A positive test result at any phase of testing indicates the need for antibody identification studies

Modified True or False.

A negative test result at any phase of testing indicates the need for antibody identification studies.

D

IgM antibodies react best at room temperature or lower and can agglutinate saline-suspended RBCs.

IgM antibodies react best at room temperature or higher and can agglutinate saline-suspended RBCs.

IgG antibodies react best at AHG phase.

---

A. Both statements are correct

B. Both statements are incorrect

C. First statement is correct, second statement is incorrect

D. First statement is incorrect, second statement is correct

• Lewis antibodies

• M antibodies

What antibodies can be a mixture of both IgG and IgM?

Autologous control

It is the patient’s RBCs tested against the patient’s serum in the same manner as the antibody screen.

A positive antibody screen and a negative autologous control indicates that an alloantibody has been detected.

Modified true or false.

A positive antibody screen and a negative autologous control indicates that an autoantibody has been detected.

True

Modified True or False.

If the patient has not been transfused within the last 3 months, a positive autologous control may indicate the presence of autoantibodies or antibodies to medications.

• Anti-Lea

• Anti-Leb

• Anti- PP1Pk

• Anti-Vel

These antibodies cause in vitro hemolysis

• Anti-Sda

• Lutheran antibodies

These antibodies cause mixed-field agglutination

• Sex

• Age

• Race

• Diagnosis

• Transfusion history

• OB history

• Medications

• IV Solutions

• Exposure to non-self RBC via transfusion or pregnancy

Antibody identification: Review History

• K

• Kpa

• Jsa

• Lua

What are the low-prevalence antigens?

• When all of the results give a negative reaction, making them not the target of the antibody

• Only if there is a homozygous expression on the antigen of the cell

When does “Ruling out” or “Rule-outs” in an interpretation happens?

Exclude antibodies that could not be responsible for the reactivity seen.

What is the first step in Exclusion?

• Do all positive cells react to the same degree?

• Do all of the positive cells react at the same phase, or do any react at different or multiple phases?

• Does the serum reactivity match any of the remaining specificities?

• Are all commonly encountered RBC antibodies excluded?

• Is the autologous control positive or negative?

• Does the patient lack the antigen corresponding to the antibody?

In the Evaluation process, what are the questions should be answered?

• Strength does/ does not indicate significance

• Influenced by dosage effect

• Presence of more that on antibody

In evaluation process, what does “Do all positive cells react to the same degree?” answers?

Indication of multiple antibodies

In evaluation process, what does “Do all of the positive cells react at the same phase, or do any react at different or multiple phases?” answers?

Testing the patient’s serum with at least 3 antigen-positive and 3 antigen-negative cells

What is the 3 and 3 rule?

When the Autologous Control is POSITIVE

When does patient’s transfusion history needs to be reviewed?

Mix-field appearance caused by alloantibodies coating the circulation donor RBCs

If the patient received transfusion within the previous 3 months, what does the positive autologous control exhibits? and is caused by?

Positive control caused by alloantibody and not by autoantibody

When autocontrol is negative, what does it indicates? and it is caused by?

Autoantibodies

The presence of these may mask the presence of alloantibodies and complicate the process of antibody identification.

Phenotyping

This is a process performed to confirm the patients’ antibody and is only performed if the patient has not been recently transfused

Coated antibodies

These needs to be removed prior to testing

Glycine EDTA

Kell antigens are denatured in this method

This needs to be incubated at room temp for 2mins

Chloroquine diphosphate

This requires 30-2hrs of incubation at room temp and 5-30mins at 37C

Negative DAT

If treated cells, this DAT result may be phenotyped.

Selected Cell Panels

This test is useful when a patient has a known antibody and the technologist attempting to determine if additional antibodies are present.

Additional Cells from different panel/s

• Ficin

• Papain

• Bromelin

• Trypsin

Identify and fill in the blanks.

Proteolytic enzymes modify the RBC surface by removing _ _ _ and by _ or removing glycoproteins.

Proteolytic enzymes

They modify the RBC surface by removing ficin, trypsin, bromelin, and papain or by removing glycoproteins.

Enzymes

Useful in identification when multiple antibodies are present

• Two-staged Method

• One-staged Method

This may be utilized instead of enhancement media:

• Enzyme treatment of panel RBCs prior to the addition of patient serum to the test tube

• Untreated panel RBC, enzyme, and patient serum are added to the test tube prior to incubation

Neutralization

This should be done to some antibodies as a way of confirmation.

This is useful when multiple antibodies are suspected and one of the suspected antibodies appears to be an antibody that can be done with this.

1. Anti-P1

2. Anti-Chido, Anti-Rodgers

3. Anti-Sda

4. Anti-Lewis

5. Anti-I

Give the antibodies of these sources of Neutralizing Substances:

1. Hydatid cyst fluid, pigeon droppings, turtledoves’ egg whites

2. Serum containing complements

3. Urine

4. Plasma or serum, saliva

5. Human breast milk

• Antibody Identification Panel

• Control (Saline and Serum)

• This is performed after neutralization using the treated serum of the patient.

• The use of this is also required to prove that loss of reactivity is due to neutralization and not to dilution of the antibody by the added substance.

• Rh

• Kidd

• Lewis

• P1

• I

• ABO

These are ENHANCED in the effect of Proteolytic Enzymes on Select Antigen-Antibody Reactions

• Duffy

• MNS

• Xga

These are INACTIVATED in the effect of Proteolytic Enzymes on Select Antigen-Antibody Reactions

Adsorption Method

In this method, Antibodies may be removed from serum by incubating the specimen with the corresponding antigen, allowing the antibody to bind to the antigen.

The antigen-antibody complex is composed of solid precipitates and is separated from the adsorbed serum by centrifugation

Human Platelet Concentrate

This is used to adsorb Bg-like antibodies from serum.

Rabbit Erythrocyte Stroma (RESt)

This possesses I, H, and IH-like structures and performs similar functions with cold reacting autoantibodies

4C

In RESt method, the patient’s serum should be incubated at what temp to remove insignificant antibodies?

RBC Adsorption

Autoantibodies are commonly removed using this technique that can detect clinically significant antibodies.

This can only be performed when the patient has not be transfused within the last 3 months.

• Patient’s RBC

• 3-6 aliquots of autologous RBCs

RBC Adsorption:

Autoadsorption process requirements

1. Allogeneic Adsorption

2. Phenotypically matched Adsorption

RBC Adsorption:

1. If autologous RBC is insufficient or if with a recent transfusion. This has the potential to adsorb an antibody to a high-prevalence antigen.

2. If the patient’s phenotype can not be determined and has not been transfused within the last 3 months and DAT-negative RBCs are available.

Antigens:

• E

• c

• K

• Jka

• s

In order for the antibodies to remain in the adsorbed serum, in Phenotypically-matched Adsorption, the donor’s cells should be negative for these antigens.

Differential Adsorption

This can be performed if the patient’s phenotype is unknown and performing antigen-typing is not an option

• R1R1

• R2R2

• rr

In Differential Adsorption method, What are the phenotype of Group O cells are used?

1. Temperature-Dependent Methods

2. 56C

3. -18C or colder, Lui Freeze-Thaw method

4. Total Elution Method

1. The simplest elution methods involve changing the temp of the antigen-antibody environment. Best at detecting IgG antibodies directed against antigens of the ABO system

2. Temp of Heat method?

3. Temp of Cold method? other name of this method?

4. This means the RBCs are destroyed by the process and can not be used for further testing.

1. Acid Elution

2. 3 pH

pH Method:

1. A common, relatively quick, and easy total elution method for the detection of Non-ABO IgG antibodies.

2. At what pH of washed antibody-coated cells mixed with glycine acid solution?

pH Method

Detection of non-ABO IgG antibodies

Disruption of antigen-antibody bond, releasing antibody into acidic supernatant

1. Dichloromethane, Xylene, Ether

2. Van der Waals forces

These organic solvents are used in total elution methods to detect non-ABO IgG antibodies.

These solvents act on the lipids in the RBC membrane to reduce surface tension and lead to the reversal of the what forces?

Organic Solvent Method

Time consuming

The chemicals used in this method pose several health and safety hazards; hence they are rarely used in the clinical lab

Antibody Titration

Quantifies the amount or concentration that is present.

To perform this, two-fold serial dilutions of serum are prepared and tested against reagent RBCs that express the target antigen.

• Flow cytometry

• Radioimmunoassay

• ELISA

Techniques that are not readily available in every laboratory under Antibody Titration.

• False

  • Two-fold serial serum dilutions are prepared and tested against reagent RBCs that express the target antigen.

• False

  • The titer level is the reciprocal of the greatest dilution in which agglutination of 1+ or greater is observed

  A score may also be assigned, based on the strength of reactivity

Modified True or False.

• Tri-fold serial dilution of serum is prepared and tested against antibodies of the RBCs that express the target antigen.

• The titer level is the reciprocal of the greatest dilution in which agglutination of 3+ or less is observed.

• False

  • The specimen should be frozen after titer

• True

• False

  • A four-fold or greater increase in titer or an increased score of 10 or more is considered to be clinically significant

Modified True or False.

• The specimen should be at room temp after determining the initial titer

• A comparison of the current specimen’s results and the initial specimen’s current results should be mad

• A four-fold or greater increase in titer or a decrease in score of 10 or less is considered to be clinically significant

Titer-level Studies

Useful in monitoring obstetric patients who have an IgG antibody that may cause HDFN

• False

  • An increase in antibody titer level during pregnancy suggests that the fetus is antigen-positive and therefore at risk of developing HDFN

• False

  • An antibody titer may also be used to help differentiate immune anti-D from passively acquired anti-D due to RhIg administration

Modified True or False.

• A decrease in antibody titer level during pregnancy suggests that the fetus is antibody-positive and therefore at risk of developing HDFN

• An antibody titer may also be used to help differentiate immune anti-D from actively acquired anti-D due to RhIg administration

Performing a Titer

Performing this is one way to confirm the presence of antibodies that have previously been know as HTLA antibodies.

• Anti-Ch

• Anti-Rg

• Anti-Csa

• Anti-Yka

• Anti-Kna

• Anti-McC

• Anti-JMH

Examples of not clinically significant antibodies but may mask antibodies that are clinically significant

• True

• True

Modified True or False.

If clinically significant:

• Units for transfusion must be antigen negative

• Must also demonstrate compatibility at AHG phase

Sickle Cell

Beta-thalassemia

Patient/s with these types of condition/s received units that are phenotypically matched even if they have not formed any alloantibodies. will also be more likely to make alloantibodies due to repeated transfusion

The most adorable human in the universe

My lovely Jasmyn Danielly Patubo Manuel