Principles of Agglutination

Precipitation and agglutination are the visible expression of the aggregation of antigens and antibodies through the formation of a framework in which antigen particles or molecules alternate with antibody molecules (Fig. 10-1). Precipitation is the term for the aggregation of soluble test antigens. Precipitation is the combination of soluble antigen with soluble antibody to produce a visible insoluble complex. Agglutination is the process whereby specific antigens (e.g., red blood cells) aggregate to form larger visible clumps when the corresponding specific antibody is present in the serum.

Artificial carrier particles may be needed to indicate visibly that an antigen-antibody reaction has taken place; examples include latex particles and colloidal charcoal. Cells unrelated to the antigen, such as erythrocytes coated with antigen in a constant amount, can be used as biological carriers. Whole bacterial cells can contain an antigen that will bind with antibodies produced in response to that antigen when it is introduced into the host (Table 10-1).

The quality of test results depends on the following technical factors:

Agglutination tests are easy to perform and, in some cases, are the most sensitive tests currently available. It is important to note that quality results are dependent on the proper training of the person performing the assay and adherence to strict quality control regulations (e.g., positive and negative control sera). Agglutination-type tests have a wide range of applications in the clinical diagnosis of noninfectious immune disorders and infectious disease.

Latex Agglutination

In latex agglutination procedures (Box 10-1), antibody molecules can be bound to the surface of latex beads. Many antibody molecules can be bound to each latex particle, increasing the potential number of exposed antigen-binding sites. If an antigen is present in a test specimen such as C-reactive protein, the antigen will bind to the combining sites of the antibody exposed on the surface of the latex beads, forming visible cross-linked aggregates of latex beads and antigen (Fig. 10-2). In some procedures (e.g., pregnancy testing, rubella antibody testing), latex particles can be coated with antigen. In the presence of serum antibodies, these particles agglutinate into large visible clumps.

Procedures based on latex agglutination must be performed under standardized conditions. The amount of antigen-antibody binding is influenced by factors such as pH, osmolarity, and ionic concentration of the solution. A variety of conditions can produce false-positive or false-negative reactions in agglutination testing (see Table 10-4).

Coagglutination and liposome-enhanced testing are variations of latex agglutination (Fig. 10-3). Coagglutination uses antibodies bound to a particle to enhance the visibility of agglutination. It is a highly specific method but may not be as sensitive as latex agglutination for detecting small quantities of antigen.

Pregnancy Testing

The principle of antigen and antibody interaction has been applied to pregnancy testing since the first agglutination tests were developed in the 1960s. These assays have replaced animal testing.

The rapid, direct, monoclonal latex slide agglutination test for detection of hCG is based on the principle of agglutination between latex particles coated with anti-hCG antibodies and hCG, if present, in the test specimen.

See for the procedural protocol.

Flocculation Tests

Flocculation tests for antibody detection are based on the interaction of soluble antigen with antibody, which results in the formation of a precipitate of fine particles. These particles are macroscopically or microscopically visible only because the precipitated product is forced to remain in a confined space.

Flocculation testing can be used in syphilis serologic testing (see Chapter 18). These tests are the classic Venereal Disease Research Laboratories (VDRL) and rapid plasma reagin (RPR) tests. In the VDRL test, an antibody-like protein, reagin, binds to the test antigen, cardiolipin-lecithin–coated cholesterol particles, and produces the particles that flocculate. In the RPR test, the antigen, cardiolipin-lecithin–coated cholesterol with choline chloride, also contains charcoal particles that allow for macroscopically visible flocculation.

Direct Bacterial Agglutination

Direct agglutination of whole pathogens can be used to detect antibodies directed against the pathogens. The most basic tests measure the antibody produced by the host to antigen determinants on the surface of a bacterial agent in response to infection with that bacterium. In a thick suspension of the bacteria, the binding of specific antibodies to surface antigens of the bacteria causes the bacteria to clump together in visible aggregates. This type of agglutination is called bacterial agglutination.

The formation of aggregates in solution is influenced by electrostatic and other forces; therefore, certain conditions are usually necessary for satisfactory results. The use of sterile physiologic saline with free positive ions in the agglutination procedure enhances the aggregation of bacteria because most bacterial surfaces exhibit a negative charge that causes them to repel each other. Because it allows more time for the antigen-antibody reaction, tube testing is considered more sensitive than slide testing. The small volume of liquid used in slide testing requires rapid reading before the liquid evaporates.

Hemagglutination

The hemagglutination method of testing detects antibodies to erythrocyte antigens. The antibody-containing specimen can be serially diluted and a suspension of red blood cells (RBCs) added to the dilutions. If a sufficient concentration of antibody is present, the erythrocytes are cross-linked and agglutinated. If nonreacting antibody or an insufficient quantity of antibody is present, the erythrocytes will fail to agglutinate.

By binding different antigens to the RBC surface in indirect hemagglutination or passive hemagglutination (PHA), the hemagglutination technique can be extended to detect antibodies to antigens other than those present on the cells (Box 10-2). Chemicals such as chromic chloride, tannic acid, and glutaraldehyde can be used to cross-link antigens to the cells.

Some antibodies (e.g., immunoglobulin G [IgG]) do not directly agglutinate erythrocytes. This incomplete or blocking type of antibody may be detected by using an enhancement medium such as antihuman globulin (AHG) reagent (also known as Coombs reagent). If AHG reagent is added, this second antibody binds to the antibody present on the erythrocytes (see procedure in Chapter 26).

Mechanisms of Agglutination

Agglutination is the clumping of particles that have antigens on their surface, such as erythrocytes, by antibody molecules that form bridges between the antigenic determinants. This is the end point for most tests involving erythrocyte antigens. Agglutination is influenced by a number of factors and is believed to occur in two stages, sensitization and lattice formation.

Sensitization

The first phase of agglutination, sensitization, represents the physical attachment of antibody molecules to antigens on the erythrocyte membrane. The combination of antigen and antibody is a reversible chemical reaction. Altering the physical conditions can result in the release of antibody from the antigen-binding site. When physical conditions are purposely manipulated to break the antigen-antibody complex, with subsequent release of the antibody into the surrounding medium, the procedure is referred to as an elution.

The amount of antibody that will react is affected by the equilibrium constant, or affinity constant, of the antibody. In most cases, the higher the equilibrium constant, the higher is the rate of association and the slower the rate of dissociation of antibody molecules. The degree of association between antigen and antibody is affected by a variety of factors and can be altered in some cases in vitro by altering some of the factors that influence antigen-antibody association, including the following:

• Particle charge

• Electrolyte concentration and viscosity

• Antibody type

• Antigen-to-antibody ratio

• Antigenic determinants

• Physical conditions (e.g., pH, temperature, duration of incubation)

Particle Charge

Inert particles such as latex, RBCs, and bacteria have a net negative surface charge called the zeta potential (Fig. 10-4). The concentration of salt in the reaction medium has an effect on antibody uptake by the membrane-bound erythrocyte antigens. Sodium (Na⁺) and chloride (Cl⁻) ions in a solution have a shielding effect. These ions cluster around and partially neutralize the opposite charges on antigen and antibody molecules, which hinders antibody-antigen association. By reducing the ionic strength of a reaction medium (e.g., using low ionic strength saline [LISS]), antibody uptake is enhanced. Charges can be overcome by centrifugation, addition of charged molecules (e.g., albumin, LISS), or enzyme pretreatment to permit the cross-linking that results in agglutination (Table 10-2).

Table 10-2

Techniques to Reduce Zeta Potential

Technique	Action
Enzyme pretreatment of red blood cells	Removes negatively charged sialic acid residues from cell surface membrane
Addition of colloids (e.g., albumin)	Increases electrical conductivity of environment
Centrifugation	Mechanical process to force red blood cells closer together

Adapted from Lehman CA: Saunders manual of clinical laboratory science, Philadelphia, 1998, WB Saunders, p 391.

Antibody Type

Immunoglobulin M (IgM) antibodies are more efficient at agglutination because their large size and multivalency permit more effective bridging of the space between cells caused by zeta potential. IgG antibodies are too small to overcome electrostatic forces between cells. The use of AHG forms cross-links between antibody molecules that have bound to the surface of RBCs. This promotes this formation of agglutination and allows for visual observation of an antigen-antibody reaction.

Antigen-Antibody Ratio

Under conditions of antibody excess, there is a surplus of molecular antigen-combining sites not bound to antigenic determinants. Precipitation reactions depend on a zone of equivalence, the zone in which optimum precipitation occurs, because the number of multivalent sites of antigens and antibodies are approximately equal. For a precipitation reaction to be detectable, the reaction must occur in the zone of equivalence. In this zone, each antibody or antigen binds to more than one antigen or antibody, respectively, forming a stable lattice or network (see later). This lattice hypothesis is based on the assumptions that each antibody molecule must have at least two binding sites and that an antigen must be multivalent.

On either side of the zone of equivalence, precipitation is prevented because of an excess of antigen or antibody. If excessive antibody concentration is present, the phenomenon known as the prozone phenomenon occurs, which can result in a false-negative reaction. In this case, antigen combines with only one or two antibody molecules and no cross-linkages are formed. This phenomenon can be overcome by serially diluting the antibody-containing serum until optimum amounts of antigen and antibody are present in the test system.

If an excess of antigen occurs, the postzone phenomenon occurs, in which small aggregates (clumps) are surrounded by excess antigen and no lattice formation is established. Excess antigen can block the presence of a small amount of antibody. To correct the postzone phenomenon, a repeat blood specimen should be collected 1 or more weeks later. If an active antibody reaction is occurring in vivo, the titer of antibody will increase and should be detectable. Repeated negative results generally suggest that the patient has the specific antibody being tested for by the procedure.

Antigenic Determinants

The placement and number of antigenic determinants both affect agglutination. For example, the A blood group antigen has more than 1.5 million sites/RBC, whereas the Kell blood group antigen has about 3500 to 6000 sites/RBC. If the number of antigenic sites is small or if the antigenic sites are buried deeply in the cell membranes, antibodies will be unable physically to contact antigenic sites.

Steric hindrance is an important physiochemical effect that influences antibody uptake by cell surface antigens. If dissimilar antibodies with approximately the same binding constant are directed against antigenic determinants located close to each other, the antibodies will compete for space in reaching their specific receptor sites. The effect of this competition can be mutual blocking, or steric hindrance, and neither antibody type will be bound to its respective antigenic determinant. Steric hindrance can occur whenever there is a conformational change in the relationship of an antigenic receptor site to the outside surface. In addition to antibody competition, competition with bound complement, other protein molecules, or the action of agents that interfere with the structural integrity of the cell surface can produce steric hindrance.

pH

The pH of the medium used for testing should be near physiologic conditions, or an optimum pH of 6.5 to 7.5. At a neutral pH, high electrolyte concentrations act to neutralize the net negative charge of particles.

Temperature and Length of Incubation

The optimum temperature needed to reach equilibrium in an antibody-antigen reaction differs for different antibodies. IgM antibodies are cold-reacting (thermal range, 4° C to 22° C [39° F to 72° F]), and IgG antibodies are warm-reacting, with an optimum temperature of reaction at 37° C (98.6° F).

The duration of incubation required to achieve maximum results depends on the rate of association and dissociation of each specific antibody. In laboratory testing, incubation times range from 15 to 60 minutes. The optimum time of incubation varies, depending on the class of immunoglobulin and how tightly an antibody attaches to its specific antigen.

Lattice Formation

Lattice formation, or the establishment of cross-links between sensitized particles (e.g., erythrocytes) and antibodies, resulting in aggregation, is a much slower process than the sensitization phase. The formation of chemical bonds and resultant lattice formation depend on the ability of a cell with attached antibody on its surface to come close enough to another cell to permit the antibody molecules to bridge the gap and combine with the antigen receptor site on the second cell. As antigens and antibodies combine, a multimolecular lattice increases in size until it precipitates out of solution as a solid particle. Cross-linking is influenced by factors such as the zeta potential.

Graded Agglutination Reactions

Observation of agglutination is initially made by gently shaking the test tube containing the serum and cells and viewing the lower portion, the button, with a magnifying glass as it is dispersed. Because agglutination is a reversible reaction, the test tube must be treated delicately, and hard shaking must be avoided; however, all the cells in the button must be resuspended before an accurate observation can be determined. Attention should also be given to whether discoloration of the fluid above the cells, the supernatant, is present. Rupture or hemolysis of erythrocytes is as important a finding as agglutination.

The strength of agglutination (Table 10-3; Fig. 10-5), called grading, uses a scale of 0, or negative (no agglutination), to 41 (all erythrocytes clumped). Table 10-4 describes false-positive and false-negative reactions. Pseudoagglutination, or the false appearance of clumping, may rarely occur because of rouleaux formation. Rouleaux formation can be encountered in patients with high or abnormal types of globulins in their blood, such as in multiple myeloma or after receiving dextran as a plasma expander. On microscopic examination, the erythrocytes appear as rolls resembling stacks of coins. To disperse the pseudoagglutination, a few drops of physiologic NaCl (saline) can be added to the reaction tube, remixed, and reexamined. This procedure, saline replacement, should be performed carefully after pseudoagglutination is suspected. It should never be done before the initial testing protocol is followed; a false-negative result may occur from the dilutional effect of the saline.

Table 10-3

Grading Agglutination Reactions

Grade	Description
Negative	No aggregates
Mixed field	A few isolated aggregates; mostly free-floating cells; supernatant appears red
Weak (±)	Tiny aggregates barely visible macroscopically; many free erythrocytes; turbid and reddish supernatant
1+	A few small aggregates just visible macroscopically; many free erythrocytes; turbid and reddish supernatant
2+	Medium-sized aggregates; some free erythrocytes; clear supernatant
3+	Several large aggregates; some free erythrocytes; clear supernatant
4+	All erythrocytes are combined into one solid aggregate; clear supernatant.

Table 10-4

Causes of False-Positive and False-Negative Agglutination Test Reactions

Cause	Correction
False-Positive Reactions
Contaminated equipment or reagents may cause particles to clump.	Store equipment and reagent in clean, dust-free environment, and handle with care. Use negative quality control (QC) steps.
Autoagglutination	Use a control with saline and no antibody as a negative control. If positive, patient’s result is invalid.
Delay in reading slide reactions results in drying out of mixture.	Follow procedural directions and read reactions exactly as specified.
Overcentrifugation causes cells or particles to clump too tightly.	Calibrate centrifuge to proper speed and time.
False-Negative Reactions
Inadequate washing of red blood cells in antihuman globulin (AHG) testing∗ may result in unbound immunoglobulins neutralizing the reagent.	Wash cells according to directions. Use positive and negative QC steps.
Failure to add AHG reagent	Use positive QC steps.
Contaminated or expired reagents	Use positive and negative QC steps.
Improper incubation	Follow procedural protocol exactly. Use positive and negative QC steps.
Delay in reading slide reactions	Follow procedural protocol exactly. Use positive and negative control steps.
Undercentrifugation	Calibrate centrifuge to proper speed and time.
Prozone phenomenon	Dilute patient serum containing antibody, and repeat the procedure.

^∗See Chapter 26.

The reverse (serum) typing procedure to confirm ABO blood grouping is based on the presence or absence of the antibodies, anti-A and anti-B, in serum. If these antibodies are present in serum, agglutination should be demonstrated when the serum is combined with reagent erythrocytes expressing A or B antigens.

Reverse typing is a cross-check for forward typing (see Chapter 2 procedure). Because of the lack of synthesized immunoglobulins in newborn and very young infants, this procedure is not performed on specimens from these patients.

See for the procedural protocol.

Chapter Highlights