Identification of Antibodies to Red Cell Antigens

Mohamed Wagih Eltonsy MBBS, M.Sc Clinical Pathologist, Blood Bank Dammam Regional Laboratory April 12, 2011

Introduction

Naturally occurring anti-A and anti-B are the only red cell antibodies that are regularly found in normal human serum or plasma. All other antibodies are called unexpected red cell antibodies

There are two types of unexpected red cell antibodies: alloantibodies and autoantibodies.
When someone produces an antibody to an antigen that he or she lacks, the antibody is called an alloantibody. When someone produces an antibody to an antigen that he or she possesses, the antibody is called an autoantibody.

Introduction

Immunization to red cell antigens may result from

pregnancy transfusion

transplantation
injections of immunogenic material In some instances, no specific immunizing event can be identified. (exposure to environmental, bacterial, or viral antigens that are similar to blood group antigens)

Introduction

Also, antibodies detected in serologic tests may be passively acquired from

Injected immunoglobulin

Donor plasma
Passenger lymphocytes in transplanted organs Hematopoietic progenitor cells (HPCs)

Significance of Alloantibodies

After an antibody has been detected, its specificity should be determined and its clinical significance assessed. Alloantibodies to red cell antigens may be detected initially in any test that uses serum or plasma or in an eluate prepared from red cells coated with alloantibody. A clinically significant red cell antibody is defined as an antibody that is frequently associated with

Hemolytic disease of the fetus and newborn (HDFN) Hemolytic transfusion reactions A notable decrease in the survival of transfused red cells

PREANALYTICAL CONSIDERATIONS

Preanalytical Considerations

Before starting antibody identification testing, consider the patients medical history.

pregnancy

Transfusion

If the patient was transfused during the past 3 months

The presence of circulating donor red cells may cause mixedfield results in antigen typing tests.

Autologous adsorption techniques must not be used because alloantibodies could be adsorbed onto donor red cells.

Preanalytical Considerations

Certain diseases have been associated with red cell antibodies

Cold agglutinin disease, Raynauds phenomenon, and infections with Mycoplasma pneumonia are often associated with anti-I. Infectious mononucleosis is sometimes associated with anti-i. Patients with paroxysmal cold hemoglobinuria (PCH), which is associated with syphilis in adults and viral infections in children, may demonstrate autoantibodies with P specificity.

Preanalytical Considerations

Warm autoantibodies often accompany

warm autoimmune hemolytic anemia (WAIHA),

systemic lupus erythematosus (SLE) multiple myeloma,

chronic lymphocytic leukemia,

lymphoma.
drug-related serologic problems include intravenous immune globulin (IVIG) and Rh immune globulin (RhIG) in antenatal and postnatal patients.

Drugs are known to cause antibody identification problems.

ANALYTICAL PHASE OF ANTIBODY IDENTIFICATIN

Analytical Phase of Antibody Identification

I. SPECIMEN REQUIREMENTS II. REAGENTS III. BASIC ANTIBODY IDENTIFICATION IV. SELECTED SEROLOGIC PROCEDURES

I. SPECIMEN REQUIREMENTS

Either serum or plasma may be used for antibody detection and identification; however, plasma is not suitable for detecting complement-activating antibodies.

A 5- to 10-mL aliquot of whole blood usually contains enough serum or plasma for identifying simple antibody specificities; more whole blood may be required for complex studies.

II. REAGENTS

1. Antibody Detection cells

Red Cells Group O red cells are commercially available and are offered as sets of either two or three bottles of single-donor red cells. Pooled antibody detection red cells (usually obtained from two different donors) may be used only when testing donor serum. Reagent red cells licensed by the Food and Drug Administration (FDA) for this purpose must express the following antigens: D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Fya, Fyb, Jka, and Jkb.

The three-cell set usually offers red cells from presumed homozygous donors with double-dose expression for the following common antigens: D, C, E, c, e, M, N, S, s, Fya, Fyb, Jka, and Jkb.

II. REAGENTS

2. Antibody Identification cells

Panels Identification of an antibody to red cell antigen(s) requires testing the serum against a panel of selected red cell samples (typically 8- 14 samples) with known antigenic composition for the major blood groups.

panel cells are group O, thereby allowing the serum of any ABO group to be tested.
To be functional, a reagent red cell panel must make it possible to identify with confidence those clinically significant alloantibodies that are most commonly encountered, such as anti-D, -E, -K, and -Fya.

II. REAGENTS

3. Antiglobulin Reagents

Most antibody detection and identification studies include an indirect antiglobulin test (IAT) phase. Either anti-IgG-specific antiglobulin reagent (AHG) or polyspecific reagent, which contains anti-IgG and anticomplement, may be used. Polyspecific reagent may detect or may detect more readily antibodies that bind complement. Complement binding may be valuable in detecting and identifying certain Kidd antibodies Many serologists prefer to use IgG- specific reagents to avoid unwanted reactivity resulting from in-vitro complement binding by cold-reactive antibodies.

II. REAGENTS

4. Enhancement Media

Although the test system may consist solely of serum and red cells. most serologists use some type of enhancement medium. Many different techniques are available, including

low-ionic-strength saline (LISS) polyethylene glycol (PEG) 22% bovine albumin column agglutination solid-phase technology

III. BASIC ANTIBODY IDENTIFICATION

1. IDENTIFICATION PANEL 2. AUTOLOGOUS CONTROL (Autocontrol) 3. PHENONTYPING AUTOLOGOUS RED CELLS

1. IDETIFICATION PANEL
i. Interpreting Results

Antibody detection results are interpreted as positive or negative according to the presence or absence of reactivity (ie, agglutination or hemolysis). Interpretation of panel results can be a more complex process combining technique, knowledge, and intuitive skills. Panel results generally include both positive and negative results, which are sometimes at different phases of testing; each result should be explained by the final conclusion.

The patients phenotype and the probability of the antibody specificity are also taken into account in the final interpretation.

1. IDETIFICATION PANEL

ii. Positive And Negative Reactions

Positive reactions can be compared to the antigen patterns of the panel cells to help assign specificity. A single alloantibody usually produces a clear pattern When there is no discernible pattern to explain the reactivity, possible explanations include multiple antibodies, dosage. Negative reactions are important because they allow tentative exclusion of antibodies to antigens expressed on the nonreactive red cells

1. IDETIFICATION PANEL

iii. Exclusion, Rule-Out, or Cross-Out

A widely used first approach to the interpretation of panel results is to exclude specificities on the basis of nonreactivity of the patients serum with red cell samples that express the antigen. If an antigen is present on the red cell sample and the serum did not react with it, the presence of the corresponding antibody may be excluded, at least tentatively. After all the antigens on that red cell sample have been crossed out, the same process is performed with the other nonreactive red cells; then additional specificities are excluded.

1. IDETIFICATION PANEL

iii. Exclusion, Rule-Out, or Cross-Out

In most cases, this process leaves a group of antibodies that have not been excluded. The pattern of reactivity for each specificity that was not excluded is compared to the pattern of reactivity obtained with the test serum. If there is a pattern that matches the test serum pattern exactly, that is most likely the specificity of the antibody in the serum. If there are remaining specificities that were not excluded, additional testing is needed to eliminate remaining possibilities and to confirm the suspected specificity.

1. IDETIFICATION PANEL
iv. Selected Cells

Selected cells are red cells that have been chosen because they express certain specific antigens and lack others. For example, if a pattern of reactive red cells exactly fits antiJka, but anti-K and anti-S were not excluded, the serum should be tested with selected red cells. Ideally, red cells with the following phenotypes should be chosen: Jk(a-), K+, S; Jk(a), K, S+; and Jk(a+), K, S. The reaction pattern with these red cells should both confirm the presence of anti-Jka and should include or exclude anti-K and anti-S.

1. IDETIFICATION PANEL
v. Probability

Conclusive antibody identification requires the serum to be

tested against a sufficient number of reagent red cell

samples

A standard approach has been to require that three

antigen-positive red cell samples react and that three

antigen-negative red cell samples fail to react for each specificity identified.

The use of two reactive and two nonreactive red cell samples is also an acceptable approach for antibody confirmation.

2. Autologous Control (Autocontrol)

The autologous control, in which serum and autologous red cells are tested under the same conditions as are serum and reagent red cells, is an important part of antibody identification. If the autocontrol is positive in the antiglobulin phase, a DAT should be performed.

If the DAT is negative, antibodies to an enhancement medium constituent or autoantibodies that react only in the enhancement medium should be considered.
Autoantibodies or drugs could explain a positive DAT; however, if the patient was recently transfused, the circulating donor red cells could be coated with alloantibody, resulting in a positive DAT, the corresponding antigen is expected to be absent from the autologous red cells.

3. Phenotyping Autologous Red Cells

It may be difficult to determine the patients phenotype if the patient was transfused in the past 3 months. A pretransfusion specimen, if available, should be used to determine the phenotype. If a pretransfusion sample is not available, the patients newly formed autologous red cells can be separated from the transfused red cells by centrifugation and then typed. New autologous red cells must be isolated from the sample while the sample is fresh. The technique is ineffective if the sample is too old (>3 days), if the patient is not producing new red cells, or if the patient has sickle-cell anemia.

3. Phenotyping Autologous Red Cells

Additional problems that may complicate red cell typing include cold and warm autoantibodies. If the cold autoantibodies are very potent, it may be necessary to treat the red cells with Dithiothreitol (DTT) to break the immunoglobulin M (IgM) molecules that cause spontaneous agglutination. When red cells are coated with IgG autoantibodies, it is not possible to perform typing that requires an IAT. However, it is often possible to type antibody-coated red cells with directly agglutinating antisera, such as IgM monoclonal reagents. Most directly agglutinating monoclonal reagents will give valid phenotyping results despite a positive DAT.

3. Phenotyping Autologous Red Cells

For antisera that require the IAT (eg, anti-Fya and anti-Fyb), it will be necessary to strip the IgG antibodies from the test red cells before typing. Common techniques for removing IgG antibodies include:

gentle heat elution

treatment with chloroquine

treatment with acid glycine/EDTA

Molecular genotyping offers an alternative to serologic typing and is especially useful in situations

where the patient has been recently transfused

or where the patients red cells are heavily coated with IgG.

IV. Selected Serologic Procedures

Many techniques and methods are used in complex antibody identification. It is important to remember that no single method is optimal for detecting all antibodies

When a pattern of weak reactions fails to indicate specificity, or when the presence of an antibody is suspected but cannot be confirmed, the use of enhancement techniques may be helpful. An autocontrol should always be included with each technique.

IV. Selected Serologic Procedures

1. LISS and PEG

Both LISS and PEG techniques are used to enhance

reactivity and to reduce incubation time.

LISS may be used to suspend test red cells for use in tube or column agglutination tests, or as an additive medium for tube or solid phase tests.

Because LISS and PEG enhance autoantibodies, their use may complicate alloantibody identification in samples that also contain autoantibodies.

IV. Selected Serologic Procedures

2. Enzymes

Ficin and papain are the most frequently used enzymes. They destroy or weaken antigens such as M, N, Fya, Fyb, Xga, JMH, Ch, and Rg. Antibodies to these antigens are expected to be nonreactive with treated red cells. Conversely, ficin-treated and papain-treated red cells show enhanced reactivity with other antibodies (eg, Rh, P, I, Kidd, and Lewis). Additional enzymes that are commonly used in immunohematology laboratories include trypsin, chymotrypsin, and pronase. In addition to enhancing the reactivity of certain antibodies, enzyme techniques may be used to separate mixtures of antibodies.

IV. Selected Serologic Procedures

3. Temperature Reduction

Some antibodies react better at room temperature or below, and their specificity may be apparent only below 22 C (eg, anti-M, -N, P1, -Lea, -Leb, -A1).

An autocontrol is especially important for tests at cold temperatures because many sera also

contain anti-I or other cold-reactive

autoantibodies.

IV. Selected Serologic Procedures

4. Increased Serum-to-Cell Ratio

Increasing the volume of serum incubated with a standard volume of red cells may enhance the reactivity of antibodies that are present in low concentrations.

Increasing the serum-to-red-cell ratio is not appropriate for tests using LISS or commercial PEG, which may contain LISS.

Tests performed in a low-ionic-strength medium require

specific proportions of serum and cells

IV. Selected Serologic Procedures

5. Increased Incubation Time

For some antibodies, a 15-minute incubation period may not be sufficient to achieve equilibrium Extending the incubation time to between 30 and 60 minutes may improve the reactivity and may help clarify the pattern of reactions. Extended incubation may be contraindicated when LISS or PEG is used.

Care must be taken to use all reagents according to the manufacturers directions.

IV. Selected Serologic Procedures

6. Alteration in pH

Lowering the pH of the test system to pH 6.5 enhances the reactivity of certain antibodies, (anti-M).

Lowering the pH, however, significantly decreases the reactivity of some antibodies.

If unbuffered saline with a pH <6.0 is used to prepare red cell suspensions or for washing in an IAT,

antibodies in the Rh, Duffy, Kidd, and MNS blood

groups may lose reactivity.

IV. Selected Serologic Procedures

7. Inhibition Techniques

Soluble forms of some blood group antigens exist in

body fluids such as saliva, urine, and plasma, or the antigens can be prepared from other sources.

Such substances can be used to inhibit the reactivity

of the corresponding antibody.

For example, if a suspected anti-P1 does not produce a definitive pattern of agglutination, the loss of

reactivity after the addition of soluble P1 substance strongly suggests that the specificity is anti-P1.

IV. Selected Serologic Procedures

8. Adsorption

Antibody can be removed from a serum sample by adsorption onto red cells that express the corresponding antigen. It may be possible to harvest the bound antibody by elution or to examine the adsorbed serum for antibody(ies) remaining after the adsorption process. Adsorption techniques are useful in the following situations:

Separating multiple antibodies present in a single serum. Removing autoantibody to permit the detection or identification of underlying alloantibodies. Removing unwanted antibody (often anti- A, anti-B, or both) from serum that contains an antibody that is suitable for reagent use.

IV. Selected Serologic Procedures

8. Adsorption

Adsorption techniques are useful in the following situations: (cont.)

Confirming the presence of specific antigens on red cells by their ability to remove antibody of corresponding specificity from previously characterized serum.
Confirming the specificity of an antibody by showing that it can be adsorbed onto red cells of only a particular blood group phenotype. To ensure that an adsorption process is complete (ie, that no unadsorbed antibody remains), it is essential to confirm that the adsorbed serum is nonreactive with a reserved sample of the adsorbing red cells that was not used for adsorption.

IV. Selected Serologic Procedures

9. Elution

Elution dissociates antibodies from sensitized red cells. Bound antibody may be released by

changing the thermodynamics of antigen- antibody reactions

neutralizing or reversing forces of attraction that hold antigenantibody complexes together,

disturbing the structure of the antigen-antibody binding site.

No single method is best for all situations.

Heat or freeze-thaw elution techniques are usually restricted to the investigation of HDFN caused by ABO incompatibility. Acid or organic solvent methods are used for elution of warmreacting auto- and alloantibodies.

IV. Selected Serologic Procedures

9. Elution

Technical factors that influence the outcome of elution procedures

Incorrect technique
Incomplete washing Dissociation of antibody before elution eg. (anti-A or M)

Elution techniques are useful for the following:

Investigation of a positive DAT. Concentration and purification of antibodies, detection of weakly expressed antigens, and identification of multiple antibody specificities. Such studies are used in conjunction with an appropriate adsorption technique. Preparation of antibody-free red cells for phenotyping or autologous adsorption studies.

IV. Selected Serologic Procedures

10. Titration

Titration studies are useful in the following situations

Prenatal studies

When the antibody has a specificity that is known to cause HDFN, the results of titration studies may contribute to the decision about performing additional procedures (eg, amniocentesis) Some antibodies that agglutinate virtually all reagent red cells may give an indication of specificity by demonstrating reactivity of different strengths with different red cell samples in titration studies. Titration results may suggest that one antibody reacts at higher dilutions than another antibody, this can allow the serum to be diluted before testing with a red cell panel, effectively removing one antibody and allowing the other to be identified.

Antibody identification and separating multiple antibodies

POSTANALYTICAL INTERPRETATION

Selecting Blood for Transfusion

After an antibody has been identified, it is important to determine its clinical significance. Antibodies that react at 37 C, by IAT, or both, are potentially clinically significant.

Antibodies that react at room temperature and below are usually not clinically significant; however, there are many exceptions.

For example, anti-Vel, -P, and -PP1Pk (-Tja) may react only at cold temperatures, yet may cause red cell destruction in-vivo. Anti-Ch, -Rg, and many of the Knops and Cost antibodies have little or no clinical significance despite their reactivity by an IAT.

Phenotyping Donor Units

Whenever possible, RBC units selected for transfusion to a patient with potentially clinically significant antibodies should be tested and found negative for the corresponding antigen(s). Even if the antibody is no longer detectable, all subsequent RBC transfusions to that patient should lack the antigen in order to prevent a secondary immune response. The transfusion service must maintain records of all patients in whom clinically significant antibodies have been previously identified An AHG- crossmatch procedure is required if the serum contains or has previously containeda clinically significant antibody.

Antigen-Negative Blood vs Crossmatch for Compatibility

For certain antibodies, typing the donor units may not be necessary, and the patients serum can be used to select serologically compatible RBC units. This is especially true for antibodies that characteristically react below 37 C (eg, anti-M, -N, -P1, -Lea, -Leb, -A1) and that do not ordinarily produce a secondary immune response following the transfusion of antigen- positive RBC units. It is rarely necessary to provide phenotypically matched antigen-negative RBC units as a prophylactic measure when the patient has no detectable antibody.

When Rare Blood Is Needed

Rare blood includes units that are negative for highprevalence antigens, as well as units that are negative for a combination of common antigens. Family members are potential source of rare blood donors. Siblings are often the best source of serologically compatible blood. The absence of high-prevalence antigens is usually associated with the inheritance of the same rare recessive blood group gene from each heterozygous parent.

When Rare Blood Is Needed

Children from the same parents have one chance in four of inheriting the same two rare genes, making siblings much more likely than the general population to express the rare type. In most cases, blood from the patients parents, children, and half of the patients siblings will express only one rare gene. If transfusion is essential and if there is no alternative to transfusing incompatible blood, these heterozygous (single-dose) donors would be preferable to random donors.

Summary

Summary