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Lab 17: Serology, Direct and Indirect Serologic Testing - Biology


DISCUSSION

A. INTRODUCTION TO SEROLOGIC TESTING

The adaptive immune responses refer to the ability of the body (self) to recognize specific foreign antigens (non-self) that threaten its biological integrity. There are two major branches of the adaptive immune responses:

1. humoral immunity: humoral immunity involves the production of antibody molecules in response to an antigen and is mediated by B-lymphocytes.

2. cell-mediated immunity: Cell-mediated immunity involves the production of cytotoxic T-lymphocytes, activated macrophages, activated NK cells, and cytokines in response to an antigen and is mediated by T-lymphocytes.

To understand the immune responses we must first understand what is meant by the term antigen. Technically, an antigen is defined as a substance that reacts with antibody molecules and antigen receptors on lymphocytes. An immunogen is an antigen that is recognized by the body as nonself and stimulates an adaptive immune response. For simplicity, both antigens and immunogens are usually referred to as antigens.

Chemically, antigens are large molecular weight proteins (including conjugated proteins such as glycoproteins, lipoproteins, and nucleoproteins) and polysaccharides (including lipopolysaccharides). These protein and polysaccharide antigens are found on the surfaces of viruses and cells, including microbial cells (bacteria, fungi, protozoans) and human cells.

As mentioned above, the B-lymphocytes and T-lymphocytes are the cells that carry out adaptive immune responses. The body recognizes an antigen as foreign when that antigen binds to the surfaces of B-lymphocytes and T-lymphocytes by way of antigen-specific receptors having a shape that corresponds to that of the antigen, similar to interlocking pieces of a puzzle. The antigen receptors on the surfaces of B-lymphocytes are antibody molecules called B-cell receptors or sIg; the receptors on the surfaces of T-lymphocytes are called T-cell receptors (TCRs).

The actual portions or fragments of an antigen that react with receptors on B-lymphocytes and T-lymphocytes, as well as with free antibody molecules, are called epitopes. The size of an epitope is generally thought to be equivalent to 5-15 amino acids or 3-4 sugar residues. Some antigens, such as polysaccharides, usually have many epitopes, but all of the same specificity. This is because polysaccharides may be composed of hundreds of sugars with branching sugar side chains, but usually contain only one or two different sugars. As a result, most "shapes" along the polysaccharide are the same (see Fig. 1). Other antigens such as proteins usually have many epitopes of different specificities. This is because proteins are usually hundreds of amino acids long and are composed of 20 different amino acids. Certain amino acids are able to interact with other amino acids in the protein chain and this causes the protein to fold over upon itself and assume a complex three-dimensional shape. As a result, there are many different "shapes" on the protein (see Fig. 2). That is why proteins are more immunogenic than polysaccharides; they are chemically more complex.

A microbe, such as a single bacterium, has many different proteins (and polysaccharides) on its surface that collectively form its various structures, and each different protein may have many different epitopes. Therefore, immune responses are directed against many different parts or epitopes of the same microbe.

Flash animation of epitopes reacting with specific sIg on
B-lymphocytes.

html5 version of animation for iPad showing epitopes reacting with specific B-cell receptor on a B-lymphocytes.

In terms of infectious diseases, the following may act as antigens:

1.Microbial structures (cell walls, capsules, flagella, pili, viral capsids, envelope-associated glycoproteins, etc.); and

2. Microbial toxins

Certain non-infectious materials may also act as antigens if they are recognized as "nonself" by the body. These include:

1. Allergens (dust, pollen, hair, foods, dander, bee venom, drugs, and other agents causing allergic reactions);

2. Foreign tissues and cells (from transplants and transfusions); and

3. The body's own cells that the body fails to recognize as "normal self" (cancer cells, infected cells, cells involved in autoimmune diseases).

Antibodies or immunoglobulins are specific protein configurations produced by B-lymphocytes and plasma cells in response to a specific antigen and capable of reacting with that antigen. Antibodies are produced in the lymphoid tissue and once produced, are found mainly in the plasma portion of the blood (the liquid fraction of the blood before clotting). Serum is the liquid fraction of the blood after clotting.

There are 5 classes of human antibodies: IgG, IgM, IgA, IgD, and IgE. The simplest antibodies, such as IgG, IgD, and IgE, are "Y"-shaped macromolecules called monomers composed of four glycoprotein chains. There are two identical heavy chains having a high molecular weight that varies with the class of antibody. In addition, there are two identical light chains of one of two varieties: kappa or gamma. The light chains have a lower molecular weight. The four glycoprotein chains are connected to one another by disulfide (S-S) bonds and noncovalent bonds (see Fig. 3A). Additional S-S bonds fold the individual glycoprotein chains into a number of distinct globular domains. The area where the top of the "Y" joins the bottom is called the hinge. This area is flexible to enable the antibody to bind to pairs of epitopes various distances apart on an antigen.

The two tips of the "Y" monomer are referred to as the Fab portions of the antibody (see Fig. 3A). The first 110 amino acids or first domain of both the heavy and light chain of the Fab region of the antibody provide specificity for binding an epitope on an antigen. The Fab portions provide specificity for binding an epitope on an antigen. The bottom part of the "Y" is called the Fc portion and this part is responsible for the biological activity of the antibody (see diagram of IgG; Fig. Depending on the class of antibody, biological activities of the Fc portion of antibodies include the ability to activate the complement pathway (IgG & IgM), bind to phagocytes (IgG, IgA), or bind to mast cells and basophils (IgE).

Two classes of antibodies are more complex. IgM is a pentamer (see Fig. 3B), consisting of 5 "Y"-like molecules connected at their Fc portions, and secretory IgA is a dimer consisting of 2 "Y"-like molecules (see Fig. 3C).

For more information on antigens, antibodies, and antibody production, see the following Learning Objects in your Lecture Guide:

  • Antigens; Unit 6, Section IA2
  • Antibody Structure; Unit 6 Section IIA2

Serology refers to using antigen-antibody reactions in the laboratory for diagnostic purposes. Its name comes from the fact that serum, the liquid portion of the blood where antibodies are found is used in testing. Serologic testing may be used in the clinical laboratory in two distinct ways:

a. To identify unknown antigens (such as microorganisms). This is called direct serologic testing. Direct serologic testing uses a preparation known antibodies, called antiserum, to identify an unknown antigen such as a microorganism.

b. To detect antibodies being made against a specific antigen in the patient's serum. This is called indirect serologic testing. Indirect serologic testing is the procedure by which antibodies in a person's serum being made by that individual against an antigen associated with a particular disease are detected using a known antigen.

Antigen-antibody reactions may be detected in the laboratory by a variety of techniques. Some of the commonly used techniques for observing in vitro antigen-antibody reactions are briefly described below.

a. Agglutination

Known antiserum causes bacteria or other particulate antigens to clump together or agglutinate. Molecular-sized antigens can be detected by attaching the known antibodies to larger, insoluble particles such as latex particles or red blood cells in order to make the agglutination visible to the naked eye.

b. Precipitation

Known antiserum is mixed with soluble test antigen and a cloudy precipitate forms at the zone of optimum antigen-antibody proportion.

c. Complement-fixation

Known antiserum is mixed with the test antigen and complement is added. Sheep red blood cells and hemolysins (antibodies that lyse the sheep red blood cells in the presence of free complement) are then added. If the complement is tied up in the first antigen-antibody reaction, it will not be available for the sheep red blood cell-hemolysin reaction and there will be no hemolysis. A negative test would result in hemolysis.

d. Enzyme-linked immunosorbant assay or ELISA (also known as Enzyme immunoassay or EIA)

Test antigens from specimens are passed through a tube (or a membrane) coated with the corresponding specific known antibodies and become trapped on the walls of the tube (or on the membrane). Known antibodies to which an enzyme has been chemically attached are then passed through the tube (or membrane) where they combine with the trapped antigens. Substrate for the attached enzyme is then added and the amount of antigen-antibody complex formed is proportional to the amount of enzyme-substrate reaction as indicated by a color change.

e. Radioactive binding techniques

Test antigens from specimens are passed through a tube coated with the corresponding specific known antibodies and become trapped on the walls of the tube. Known antibodies to which a radioactive isotope has been chemically attached are then passed through the tube where they combine with the trapped antigens. The amount of antigen-antibody complex formed is proportional to the degree of radioactivity.

f. Fluorescent antibody technique

A fluorescent dye is chemically attached to the known antibodies. When the fluorescent antibody reacts with the antigen, the antigen will fluoresce when viewed with a fluorescent microscope.

B. DIRECT SEROLOGIC TESTING: USING ANTIGEN-ANTIBODY REACTIONS IN THE LABORATORY TO IDENTIFY UNKNOWN ANTIGENS SUCH AS MICROORGANISMS.

This type of serologic testing employs known antiserum (serum containing specific known antibodies). The preparation of known antibodies is prepared in one of two ways: in animals or by hybridoma cells.

1. Preparation of known antisera in animals.

Preparation of known antiserum in animals involves inoculating animals with specific known antigens such as a specific strain of a bacterium. After the animal's immune responses have had time to produce antibodies against that antigen, the animal is bled and the blood is allowed to clot. The resulting liquid portion of the blood is the serum and it will contain antibodies specific for the injected antigen.

However, one of the problems of using antibodies prepared in animals (by injecting the animal with a specific antigen and collecting the serum after antibodies are produced) is that up to 90% of the antibodies in the animal's serum may be antibodies the animal has made "on its own" against environmental antigens, rather than those made against the injected antigen. The development of monoclonal antibody technique has largely solved that problem.

2. Preparation of known antibodies by monoclonal antibody technique.

One of the major breakthroughs in immunology occurred when monoclonal antibody technique was developed. Monoclonal antibodies are antibodies of a single specific type. In this technique, an animal is injected with the specific antigen (see Fig. 4, step 1) for the antibody desired. After appropriate time for antibody production, the animal's spleen is removed. The spleen is rich in plasma cells and each plasma cell produces only one specific type of antibody. However, plasma cells will not grow artificially in cell culture. Therefore, a plasma cell producing the desired antibody is fused with a myeloma cell ,a cancer cell from bone marrow which will grow rapidly in cell culture, to produce a hybridoma cell (see Fig. 4, step 2). The hybridoma cell has the characteristics of both parent cells. It will produce the specific antibodies like the plasma cell and will also grow readily in cell culture like the myeloma cell. The hybridoma cells are grown artificially in huge vats where they produce large quantities of the specific antibody (see Fig. 4, step 3).

Monoclonal antibodies are now used routinely in medical research and diagnostic serology and are being used experimentally in treating certain cancers and a few other diseases.

3. The concept and general procedure for direct serologic testing.

The concept and general procedure for using antigen-antibody reactions to identify unknown antigens are as follows:

  • Concept:

    This testing is based on the fact that antigen- antibody reactions are very specific. Antibodies usually react only with the antigen that stimulated their production in the first place, and are just as specific as an enzyme-substrate reaction. Because of this, one can use known antiserum (prepared by animal inoculation or monoclonal antibody technique as discussed above) to identify unknown antigens such as a microorganism.

  • General Procedure:

    A suspension of the unknown antigen to be identified is mixed with known antiserum for that antigen. One then looks for an antigen-antibody reaction.

Examples of serologic tests used to identify unknown microorganisms include the serological typing of Shigella and Salmonella (Lab 13), the Lancefield typing of beta streptococci (Lab 14), and the serological identification of Neisseria gonorrhoeae and Neisseria meningitidis (Lab 16). Serological tests used to identify antigens which are not microorganisms include blood typing, tissue typing, and pregnancy testing.

4. Examples of direct serologic testing to identify unknown antigens

As stated above, this type of serologic testing uses known antiserum (antibodies) to identify unknown antigens. Four such tests will be looked at in lab today.

a. Serological Typing of Shigella

There are four different serological subgroups of Shigella, each corresponding to a different species:

  • subgroup A = Shigella dysenteriae
  • subgroup B = Shigella flexneri
  • subgroup C = Shigella boydii
  • subgroup D = Shigella sonnei

Known antiserum is available for each of the 4 subgroups of Shigella listed above and contains antibodies against the cell wall ("O" antigens) of Shigella. The suspected Shigella (the unknown antigen) is placed in each of 4 circles on a slide and a different known antiserum (A, B, C or D) is then added to each circle. A positive antigen-antibody reaction appears as a clumping or agglutination of the Shigella (see Fig. 5).

b. Serological Typing of Streptococci

The Clearview® Strep A Exact II Dipstick is a qualitative serologic test for detecting Group A Streptococcal antigen (the unknown antigen) directly from throat swabs and is used as an aid in diagnosing streptococcal pharyngitis caused by Streptococcus pyogenes (Group A Beta Streptococci).

The test consists of a membrane strip that is precoated with rabbit anti-Strep A antibody-red latex conjugate (known antibody with red latex particles attached) located in a pad at the beginning of the strip. It is also precoated with rabbit anti-Strep A antibody (known antibody without attached red latex) that is immobilized at the test line where the test results are read (see Fig. 6A). The red latex particles attached to the rabbit anti-Strep A antibody is what ultimately causes the “positive” red band.

When the test strip is immersed in the extracted sample, the Group A Streptococcal antigen extracted from the Streptococcus pyogenes on the throat swab of a person with strep throat begins to move chromatographically up the membrane and binds to the red-colored known antibody-latex conjugate in the pad located at the beginning of the strip, forming a Strep A antigen-antibody complex (see Fig. 6B). This Strep A antigen-antibody complex continues to moves up the membrane to the test line region where the immobilized rabbit anti-Strep A antibodies are located.

If Group A Streptococcal antigen is present in the throat swab, a red-colored sandwich of antibody/Strep A antigen/red latex conjugate antibody forms in the test line region of the strip (see Fig. 6C). The red color at the control line region appears when enough reagent has reached the control area and indicates that the test is finished. As a result, a positive test for Group A Strep antigen appears as a red band in the test result area and a red band in the control area (see Fig. 6C).

If there is no Group A Streptococcal antigen present in the throat swab no red band appears in the test result region of the strip and a single red band appears in the control line region, indicating a negative test for Group A Strep antigen (see Fig. 6D).

Flash Animation showing serologic identification of
Group A Streptococci, part-1.

http5 version of animation for iPad showing serologic identification of
Group A Streptococci, part-1.

Flash Animation showing serologic identification of
Group A Streptococci, part-2.

http5 version of animation for iPad showing serologic identification of
Group A Streptococci, part-2.

c. Serological Testing to Diagnose Pregnancy

The Alere® hCG Dipstick is a qualitative serologic test for detecting early pregnancy. The hormone human chorionic gonadotropin (hCG), produced by the placenta, appears in the serum and urine of pregnant females. The hCG is composed of two subunits - alpha and beta. The Alere® hCG Dipstick is a one step pregnancy test that detects levels of hCG as low as 25 mlU/ml. Human chorionic gonadotropin (hCG), the unknown antigen for which one is testing, is identified in the urine by using known mouse monoclonal antibodies against the beta subunit of hCG bound to colloidal gold, which is red in color. It also uses known goat polyclonal antibodies against the alpha subunit of hCG which is bound to the test result region of the dipstick.

Like the Strep A test mentioned above, this test uses a color immunochromatographic assay to detect the antigen-antibody reaction.The test consists of a membrane strip that is precoated with known mouse anti- beta hCG antibody-colloidal gold conjugate (known antibody with red colloidal gold particles attached) located in a pad at the beginning of the strip. It is also precoated with known goat anti-alpha hCG antibody (known antibody without attached red colloidal gold) that is immobilized at the test line where the test results are read (see Fig. 7B1). The red colloidal gold particles attached to the mouse anti-alpha hCG antibody is what ultimately causes the “positive” red band.

When the test strip is immersed in the urine sample, the hCG begins to move chromatographically up the membrane and binds to the red-colored known anti-beta hCG antibody-gold conjugate in the pad located at the beginning of the strip, forming a hCG antigen-antibody complex (see Fig. 7B2). This hCG antigen-antibody complex continues to moves up the membrane to the test line region where the immobilizedknown goat anti-beta hCG antibodies are bound.

If hCG is present in the urine, a red-colored sandwich of anti-beta antibody/hCG antigen/red gold conjugate anti-alpha antibody forms in the test line region of the strip (see Fig. 7B3). As a result, a positive test for hCG antigen appears as a red band in the test result area and a red band in the control area (see Fig. 7B3).

If there is no detectable hCG antigen present in the urine no red band appears in the test result region of the strip and a single red band appears in the control line region, indicating a negative test for hCG antigen (see Fig. 7B4).

Flash Animation showing serologic identification of
hCG, part-1.

http5 version of animation for iPad showing serologic identification of
hCG, part-1.

Flash Animation showing serologic identification of
hCG, part-2.

http5 version of animation for iPad showing serologic identification of
hCG, part-2.

d. Identification of Microorganisms Using the Direct Fluorescent Antibody Technique

Certain fluorescent dyes can be chemically attached to the known antibody molecules in antiserum. The known fluorescent antibody is then mixed with the unknown antigen,such as a microorganism, fixed to a slide. After washing, to remove any fluorescent antibody not bound to the antigen, the slide is viewed with a fluorescent microscope.

If the fluorescent antibody reacted with the unknown antigen, the antigen will glow or fluoresce under the fluorescent microscope. If the antibody did not react with the antigen, the antibodies will be washed off the slide and the antigen will not fluoresce.

Flash Animation showing a positive direct fluorescent antibody test.

http5 version of animation for iPad showing a positive direct fluorescent antibody test.

Flash Animation showing a negative direct fluorescent antibody test.

http5 version of animation for iPad showing a negative direct fluorescent antibody test.

For example, in the direct fluorescent antibody test for Neisseria gonorrhoeae, mentioned briefly in lab 16, the unknown antigen, suspected Neisseria gonorrhoeae,is fixed to a microscope slide. Known fluorescent antibodies made against N. gonorrhoeae are then added (see Fig. 8, step 1) and the slide is then washed to remove any fluorescent antibody not bound to the antigen. The slide is then viewed under a fluorescent microscope.

If the unknown antigen is Neisseria gonorrhoeae, the known antibodies against N. gonorrhoeae with attached fluorescent dye will bind to the bacterium and will not wash off. The bacteria will fluoresce when viewed under a fluorescent microscope (see Fig. 8, step 2 and Fig. 10). If the unknown antigen is not N. gonorrhoeae, the known fluorescent antibodies against will wash off the slide and the bacteria will not fluoresce when viewed under a fluorescent microscope.

Many bacteria, viruses, and fungi can be identified using this technique.

C. INDIRECT SEROLOGIC TESTING: USING ANTIGEN-ANTIBODY REACTIONS IN THE LABORATORY TO INDIRECTLY DIAGNOSE DISEASE BY DETECTING ANTIBODIES IN A PERSON'S SERUM PRODUCED AGAINST A DISEASE ANTIGEN

Indirect serologic testing is the procedure whereby antibodies in a person's serum being made by that individual against an antigen associated with a particular disease are detected using a known antigen.

1. The concept and general procedure for indirect serologic testing.

The concept and general procedure for this type of serological testing are as follows:

  • Concept:

    This type of testing is based on the fact that antibodies are only produced in response to a specific antigen. In other words, a person will not be producing antibodies against a disease antigen unless that antigen is in the body stimulating antibody production.

  • General Procedure:

    A sample of the patient's serum (the liquid portion of the blood after clotting and containing antibodies against the disease antigen if the person has or has had the disease) is mixed with the known antigen for that suspected disease. One then looks for an antigen-antibody reaction.

    Examples of serologic tests to diagnose disease by the detection of antibodies in the patient's serum include the various serological tests for syphilis or STS (such as the RPR, the VDRL, and the FTA-ABS tests), the tests for infectious mononucleosis, the tests for the Human Immunodeficiency Virus (HIV), the tests for systemic lupus erythematosus, and tests for variety of other viral infections.

2. Qualitative and quantitative serologic tests.

Indirect serologic tests may be qualitative or quantitative. A qualitative test only detects the presence or absence of specific antibodies in the patient's serum and is often used for screening purposes. A quantitative test gives the titer or amount of that antibody in the serum. Titer indicates how far you can dilute the patient's serum and still have it contain enough antibodies to give a detectable antigen-antibody reaction. In other words, the more antibodies being produced by the body, the more you can dilute the person's serum and still see a reaction. Quantitative serological tests are often used to follow the progress of a disease by looking for a rise and subsequent drop in antibody titer.

3. Examples of indirect serologic tests to detect antibodies in the patient's serum

a. The RPR Test for Syphilis

Syphilis is a sexually transmitted disease caused by the spirochete Treponema pallidum. The RPR (Rapid Plasma Reagin) Card® test is a presumptive serologic screening test for syphilis. The serum of a person with syphilis contains a non-specific anti-lipid antibody (traditionally termed reagin), which is not found in normal serum. The exact nature of the anti-lipid (reagin) antibody is not known but it is thought that a syphilis infection instigates the breakdown of the patient's own tissue cells. Fatty substances which are released then combine with protein from Treponema pallidum to form an antigen which stimulates the body to produce antibodies against both the body's tissue lipids (non-specific or non-treponemal) as well as the T. pallidum protein (specific or treponemal). The RPR Card® test detects the nonspecific antilipid antibody and is referred to as a non-treponemal test for syphilis.

- scanning electron micrograph of the spirochete Treponema pallidum; courtesy of CDC.

It must be remembered that tests for the presence of these nonspecific antilipid antibodies are meant as a presumptive screening test for syphilis. Similar reagin-like antibodies may also be present as a result of other diseases such as malaria, leprosy, infectious mononucleosis, systemic lupus erythematosus, viral pneumonia, measles, and collagen diseases and may give biologic false-positive results (BFP). Confirming tests should be made for the presence of specific antibodies against the T. pallidum itself. The confirming test for syphilis is the FTA-ABS test discussed below. Any serologic test for syphilis is referred to commonly as an STS (Serological Test for Syphilis).

The known RPR antigen consists of cardiolipin, lecithin, and cholesterol bound to charcoal particles in order to make the reaction visible to the naked eye. If the patient has syphilis, the antilipid antibodies in his or her serum will cross-react with the known RPR lipid antigens giving a visible clumping of the charcoal particles (see Fig. 1).

We will do a quantitative RPR Card® test today in lab. Keep in mind that a quantitative test allows one to determine the titer or amount of a certain antibody in the serum. In this test, a constant amount of RPR antigen is added to dilutions of the patient's serum. The most dilute sample of the patient's serum still containing enough antibodies to give a visible antigen-antibody reaction is reported as the titer.

b. Serologic Tests for Infectious Mononucleosis

During the course of infectious mononucleosis, caused by the Epstein-Barr virus (EBV), the body produces nonspecific heterophile antibodies which are not found in normal serum. As it turns out, these heterophile antibodies will cross react with glycoprotein antigens found on the surface of red blood cells (RBCs) of various animals, including horses, sheep, and cows, causing the RBCs to agglutinate. These cross-reacting glycoprotein antigens are often called Paul-Bunnell antigens after their discoverers.

The infectious mononucleosis serologic test demonstrated today is a rapid qualitative test for infectious mononucleosis that uses Paul-Bunnell antigens adsorbed to microscopic white latex particles as the "known antigen." These antigens bind specifically to the heterophile antibodies found in the serum of people with infectious mononucleosis causing the latex particles to clump or agglutinate (see Fig. Quantitative tests may then be done to determine the titer of heterophile antibodies and follow the progress of the disease.

c. Serologic Tests for Systemic Lupus Erythematosus (SLE)

Systemic lupus erythematosus or SLE is a systemic autoimmune disease. Immune complexes become deposited between the dermis and the epidermis, and in joints, blood vessels, glomeruli of the kidneys, and the central nervous system. It is four times more common in women than in men. In SLE, autoantibodies are made against components of DNA. This test is specific for the serum anti-deoxyribonucleoprotein antibodies associated with SLE. The known antigen is deoxyribonucleoprotein adsorbed to latex particles to make the reaction more visible to the eye (see Fig. 3). This is a qualitative test used to screen for the presence of the disease and to monitor its course.

d. Detecting Antibody Using the Indirect Fluorescent Antibody Technique: The FTA-ABS test for syphilis

The indirect fluorescent antibody technique involves three different reagents:

a. The patient's serum (containing antibodies against the disease antigen if the disease is present)

b. Known antigen for the suspected disease

c. Fluorescent anti-human gamma globulin antibodies (antibodies made in another animal against the Fc portion of human antibodies (see Fig. 9) by injecting an animal with human serum. A fluorescent dye is then chemically attached to the anti-human gamma globulin (anti-HGG) antibodies.

The FTA-ABS test (Fluorescent Treponemal Antibody Absorption Test) for syphilis is an example of an indirect fluorescent antibody procedure. This is a confirming test for syphilis since it tests specifically for antibodies in the patient's serum made in response to the syphilis spirochete, Treponema pallidum.

In this test, killed T. pallidum,(the known antigen), is fixed on a slide (see Fig 4, step 1). The patient's serum is added to the slide. If the patient has syphilis, antibodies against the T. pallidum will react with the antigen on the slide (Fig. The slide is then washed to remove any antibodies not bound to the spirochete.

To make this reaction visible, a second animal-derived antibody made against human antibodies and labelled with a fluorescent dye (fluorescent anti-human gamma globulin) is added. These fluorescent anti-HGG antibodies react with the patient's antibodies which have reacted with the T. pallidum on the slide (Fig. 4, step 3). The slide is washed to remove any unbound fluorescent anti-HGG antibodies and observed with a fluorescent microscope. If the spirochetes glow or fluoresce (see Fig. 5), the patient has made antibodies against T. pallidum and has syphilis.

Flash animation of the FTA-ABS test for syphilis.

http5 version of animation for iPad showing the FTA-ABS test for syphilis.

Another example of the indirect fluorescent antibody test is the test for antibodies against the measles virus. Inactivated measles virus-infected cells (the known antigen) are fixed to a microscope slide. The patient's serum is then added. If the person has measles, antibodies of the isotype IgG will be made against the measles virus and will bind to viral epitopes on the know measles virus-infected cells. After washing the slide to remove any unbound IgG, fluorescent antihuman IgG is added. The fluoprescent antihuman IgG then binds to the patient's IgG that is bound to the infected cells. When viewed with a fluorescent microscope, the infected cells will fluoresce green.

e. The EIA and Western Blot serologic tests for antibodies against the Human Immunodeficiency Virus (HIV)

In the case of the current HIV antibody tests, the patient's serum is mixed with various HIV antigens produced by recombinant DNA technology. If the person is seropositive (has repeated positive antigen-antibody tests), then HIV must be in that person's body stimulating antibody production. In other words, the person must be infected with HIV. The two most common tests currently used to detect antibodies against HIV are the enzyme immunoassay or EIA (also known as the enzyme-linked immunosorbant assay or ELISA) and the Western blot or WB. A person is considered to be seropositive for HIV infection only after an EIA screening test is repeatedly reactive and another test such as the WB has been performed to confirm the results.

The EIA is less expensive, faster, and technically less complicated than the WB and is the procedure initially done as a screening test for HIV infection. The various EIA tests give a spectrophotometric reading of the amount of antibody binding to known HIV antigens.

The EIA test kit contains plastic wells to which various HIV antigens have been adsorbed (see Fig. 6, step 1). The patient's serum is added to the wells and any antibodies present in the serum against HIV antigens will bind to the corresponding antigens in the wells (Fig. 6, step 2). The wells are then washed to remove all antibodies in the serum other than those bound to HIV antigens. Enzyme-linked anti-human gamma globulin (anti HGG) antibodies are then added to the wells. These antibodies, made in another animal against the Fc portion of human antibodies by injecting the animal with human serum, have an enzyme chemically attached. They react with the human antibodies bound to the known HIV antigens (Fig. 6, step 3). The wells are then washed to remove any anti-HGG that has not bound to serum antibodies. A substrate specific for the enzyme is then added and the resulting enzyme-substrate reaction causes a color change in the wells (Fig. 6, step 4). If there are no antibodies in the patient's serum against HIV, there will be nothing for the enzyme-linked anti-HGG to bind to and it will be washed from the wells. When the substrate is added, there will be no enzyme present in the wells to give a color change.

Flash animation of the EIA test for antibodies against HIV.

http5 version of animation for iPad showing the EIA test for antibodies against HIV.

If the initial EIA is reactive it is automatically repeated to reduce the possibility that technical laboratory error caused the reactive result. If the EIA is still reactive, it is then confirmed by the Western blot test.

The Western blot WB is the test most commonly used as a confirming test if the EIA is repeatedly positive. The WB is technically more complex to perform and interpret, is more time consuming, and is more expensive than the EIAs.

With the WB, the various protein and glycoprotein antigens from HIV are separated according to their molecular weight by gel electrophoresis (a procedure that separates charged proteins in a gel by applying an electric field). Once separated, the various HIV antigens are transferred to a nitrocellulose strip (see Fig. 7, step 1 and Fig. 7, step 2). The patient's serum is then incubated with the strip and any HIV antibodies that are present will bind to the corresponding known HIV antigens on the strip (Fig. 7, step 3). Enzyme-linked anti-human gamma globulin (anti HGG) antibodies are then added to the strip. 7, step 4). The strip is then washed to remove any anti-HGG that has not bound to serum antibodies. A substrate specific for the enzyme is then added and the resulting enzyme-substrate reaction causes a color change on the strip (Fig. 7, step 5). If there are no antibodies in the patient's serum against HIV, there will be nothing for the enzyme-linked anti-HGG to bind to and it will be washed from the strip. When the substrate is added, there will be no enzyme present on the strip to give a color change.

Flash animation of the WB test for antibodies against HIV.

http5 version of animation for iPad showing the WB test for antibodies against HIV.

It should be mentioned that all serologic tests are capable of giving occasional false-positive and false-negative results. The most common cause of a false-negative HIV antibody test is when a person has been only recently infected with HIV and his or her body has not yet made sufficient quantities of antibodies to give a visible positive serologic test. It generally takes between 2 weeks and 3 months after a person is initially infected with HIV to convert to a positive HIV antibody test.

A number of commercial rapid HIV tests have also been improved for detecting antibody against HIV. They include:

  • OraQuick Advance HIV1/2®: uses either a finger-stick blood speciman or an oral speciman.
  • Uni-Gold Recombigen®: uses either a finger-stick or whole blood speciman.
  • Reveal G2®: Uses serum or plasma.
  • Multispot HIV-1/HIV-2®: Uses serum or Plasma.

For more information on HIV and AIDS, see the following Learning Objects in your Lecture Guide:

  • Unit 4, Section IVF3: The Life Cycle of HIV

PROCEDURE

PROCEDURE FOR DIRECT SEROLOGIC TESTING TO DETECT UNKNOWN ANTIGENS

A. Serologic Typing of Shigella

1. Using a wax marker, draw two circles (about the size of a nickel) on each of two clean glass slides . Label the circles A, B, C, and D.

2. Add one drop of the suspected Shigella(unknown antigen) to each circle. (The Shigella has been treated with formalin to make it noninfectious but still antigenic.)

3. Now add one drop of known Shigella subgroup A antiserum to the "A" circle, one drop of known Shigella subgroup B antiserum to the "B" circle, one drop of known Shigella subgroup C antiserum to the "C" circle, and one drop of known Shigella subgroup D antiserum to the "D" circle.

4. Rotate the slide carefully for 30-60 seconds.

Agglutination of the bacteria, indicates a positive reaction.

No agglutination is negative.

5. Dispose of all pipettes and slides in the disinfectant container.

B. Serologic Typing of Streptococci: The Clearview® Strep A Exact II Dipstick Test

1. Add 4 drops of Extraction Reagent #1 to the extraction tube. This reagent contains 2M sodium nitrite and should be pink to purple in color.

2. Add 4 drops of Extraction Reagent #2 to the extraction tube. This reagent contains 0.3M acetic acid. The solution must turn yellow in color.

3. Place the throat swab in the extraction tube and roll it with a circular motion inside the tube. Let stand for at least 1 minute.

4. Squeeze the swab firmly against the extraction tube to expel as much liquid as possible from the swab and discard the swab in the biowaste container.

5. Immerse the test strip into the extraction tube with the arrows pointing toward the extracted sample solution. Leave the strip in the tube and start timing.

6. Read results in 5 minutes. A red band in the control region and a red band in the test region indicates a positive test (See Fig. A single red band in the control region only indicates a negative test (See Fig. 6D). No colored band in the control region indicates an invalid test.

C. Serologic Testing to Detect Pregnancy: The Alere hCG-Dipstick®

1. Dip the hCG dipstick into the urine up to the maximum line on the strip for 5 seconds.

2. Place the test dipstick on a flat, non-absorbant surface and read the results at 3-4 minutes. Do not interpret after the appropriate read time.

3. If hCG is present in the urine at a concentration of 25mlU/ml or greater, a positive test, a pink-to-red Test line will appear along with a red Control line in the Result Window (see Fig. If hCG is absent or present at very low levels, a negative test, only a red Control line appears in the Result Window (see Fig. 7B4).

D. The Direct Fluorescent Antibody Technique

Observe the demonstration of a positive direct fluorescent antibody test for Neisseria gonorrhoeae .

PROCEDURE FOR INDIRECT SEROLOGIC TESTING TO DETECT ANTIBODIES IN THE PATIENT'S SERUM

A. The RPR® Card Test for Syphilis (demonstration)

1. Label 6 test tubes as follows: 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32.

2. Using a 1.0 ml pipette, add 0.5 ml of 0.9% saline solution into tubes 1:2, 1:4, 1:8, 1:16, and 1:32.

3. Add 0.5 ml of the patient's serum to the 1:1 tube (undiluted serum).

4. Add another 0.5 ml of serum to the saline in the 1:2 tube and mix. Remove 0.5 ml from the 1:2 tube and add it to the 1:4 tube and mix. Remove 0.5 ml from the 1:4 tube, add to the 1:8 tube and mix. Remove 0.5 ml from the 1:8 tube, add to the 1:16 tube and mix. Remove 0.5 ml from the 1:16 tube, add to the 1:32 tube and mix. Remove 0.5 ml from the 1:32 tube and discard. The dilution of the serum is summarized in Fig. 8.

5. Using the capillary pipettes provided with the kit, add a drop of each serum dilution to separate circles of the RPR card. Spread the serum over the entire inner surface of the circle with the tip of the pipette, using a new pipette for each serum dilution.

6. Using the RPR antigen dispenser, add a drop of known RPR antigen to each circle. Do not let the needle of the dispenser touch the serum. Using disposable stirrers, mix the known RPR antigen with the serum in each circle.

7. Place the slide on a shaker and rotate for a maximum of 4 minutes.

8. Read the results as follows:

  • A definite clumping of the charcoal particles is reported as reactive (R).
  • No clumping is reported as non-reactive (N).

The greatest serum dilution that produces a reactive result is the titer. For example, if the dilutions turned out as follows, the titer would be reported as 1:4 or 4 dils.

1:11:21:41:81:161:32

R

R

R

N

N

N

B. The Serologic Tests for Infectious Mononucleosis (demonstration)

1. Place one drop of each of the patient's serum in circles on the test slide.

2. Add one drop of the treated latex particles containing Paul-Bunnell antigens on their surface (the known antigen) to each circle and mix with disposable applicator sticks.

3. Rock the card gently for 3 minutes,and observe for agglutination of the latex particles. Agglutination indicates the presence of heterophile antibodies (see Fig. 2).

C. The Serologic Tests for Systemic Lupus Erythematosus (SLE) (demonstration)

1. Add one drop of each of the patient's serum to separate circles on the test slide.

2. Add one drop of the Latex-Deoxyribonucleoprotein reagent (the known antigen, deoxyribonucleoprotein adsorbed to latex particles) to each serum sample and mix with disposable applicator sticks.

3. Rock the slide gently for 3 minutes and look for agglutination of the latex particles. Agglutination indicates the presence of antinuclear antibodies associated with SLE (see Fig. 3).

D. The FTA-ABS Test for Syphilis (Indirect Fluorescent Antibody Technique

Observe the 35mm slide of a positive FTA-ABS test (see Fig. 5).

E. The EIA and WB Tests for HIV Antibodies

Observe the illustrations of the EIA and the WB tests for antibodies against HIV.

RESULTS

RESULTS FOR DIRECT SEROLOGIC TESTING TO DETECT UNKNOWN ANTIGENS

A. Serologic Typing of Shigella

Make a drawing of your results.

  • Agglutination of bacteria is positive.
  • No agglutination of bacteria is negative.

Shigella typing slide

B. Serologic Typing of Streptococci: Clearview® Strep A Exact II Dipstick

Make a drawing of your results.

C. Serologic Testing to Diagnose Pregnancy: Alere® hCG Dipstick

Make a drawing of a positive test for pregnancy.

D. The Direct Fluorescent Antibody Technique

Make a drawing and describe a positive direct fluorescent antibody test.

Positive direct fluorescent antibody test
for Neisseria gonorrhoeae

RESULTS FOR INDIRECT SEROLOGIC TESTING TO DETECT ANTIBODIES IN THE PATIENT'S SERUM

A. RPR Card® Test for Syphilis (Quantitative)

Detects nontreponemal antilipid antibodies (reagin)

Record your results in the table.

DilutionResult
1:1
1:2
1:4
1:8
1:16
1:32
Titer

R = reactive (distinct clumps)
N = nonreactive (no clumps)

B. MONO-TEST for Infectious Mononucleosis (Qualitative)

Detects heterophile antibodies.

Draw the results of a positive and a negative test.

Infectious mononucleosis test slide
+ = Agglutination of latex particles
- = No agglutination of latex particles

C. Serologic test for SLE (Qualitative)

Detects anti-deoxyribonucleoprotein antibodies.

Draw the results of a positive and a negative test.

SLE test slide
+ = Agglutination
- = No agglutination

D. FTA-ABS Test for Syphilis (Confirming)

Detects antibodies against Treponema pallidum

Draw the results of a positive FTA-ABS test.

Positive FTA-ABS test for syphilis
(fluorescent spirochetes)


Lab 17: Serology, Direct and Indirect Serologic Testing - Biology

Over the last several years, intravenous IgG (IVIgG) has acquired great importance in the treatment of immunodeficiency states and autoimmune disorders, in the prophylaxis of Rh isoimmunization, and in the prophylaxis of bone marrow transplant patients to decrease infections (1-3). It is important to realize though that there are clinical laboratory testing problems associated with the use of IVIgG. These problems include those caused by viral and red blood cell (RBC) alloantibody contamination of IVIgG preparations (Table 1).

Table 1. Antibodies That May Be Present in IVIgG Preparations
TetanusAnti-A and anti-B
MeaslesAnti-A
Hepatitis B coreAnti-B
CytomegalovirusAnti-D
Hepatitis AAnti-K
Hepatitis B surface antigenAntibody (antibodies) that react with all RBC tested
Hepatitis CPlatelet antibodies (?)
Many other antibodies to bacteria, virus, etc.Lymphocytotoxic antibodies (?)

Preparations of IVIgG are extracted from large pools of volunteer plasma donors (greater than 10,000 donors/pool) under strict U.S. Food and Drug Administration (FDA) regulations and World Health Organization (WHO) standards and guidelines. IVIgG is extracted by a process of cold alcohol fractionation the final product has 96-98% of the electrophoretic characteristics of gammaglobulin. Manufacturers add various amounts of biochemical compounds to achieve isotonicity, specific pH ranges, stability, and solubility (4-16). IVIgG also contains variable amounts of IgA (from 0.4 to 720 mg/mL, depending on the manufacturer). This is important to keep in mind when treating patients who may be IgA deficient (3).

After infusion, IVIgG becomes available in the circulation almost immediately. It is estimated that after 6 d, the extravascular and intravascular compartment concentrations of IVIgG are equalized. Although the physiologic half-life of IVIgG is approximately 22 d, it has been observed to extend to over 30 d in immunodeficient patients (13). Again, IVIgG prepared by different manufacturers must comply with strict WHO and FDA guidelines, production standards, and quality controls, which include having expected minimum levels of antibodies to hepatitis B surface antigen (HBsAg) and hepatitis A virus (4).

IVIgG preparations contain the various IgG subclasses in proportions similar to those in normal plasma. The importance of the IgG subclasses in replacement therapy is significant, as many disease and infection processes seem to be associated with deficiencies in specific IgG subclasses (17-23). However, IVIgG preparations derived from the large plasma donor pools described above also contain a broad spectrum of antibodies to viral, bacterial, and other microorganisms. In addition, they unavoidably contain antibodies against RBC antigens, which may produce false-positive results during workup of transfusion-dependent patients (24) (Table 1).

Consequently, the presence of alloantibodies in IVIgG preparations is a concern because of the havoc it can wreak on serologic and compatibility test results. One of the most obvious concerns is contamination with antibodies to human immunodeficiency virus (HIV) and hepatitis C virus. We here at M. D. Anderson Cancer Center's Transfusion Service recently studied IVIgG preparations used by our service. In a study reported in 1991, we tested 165 lots of IVIgG from different manufacturers (24) (Table 2). In a more recent study, we analyzed 51 lots of IVIgG (unpublished data). Definite decreases in contamination levels could be seen when comparing our two sets of data. For instance, unlike the earlier lots, none of the current lots was reactive to human immunodeficiency virus 1/2 (HIV-1/2) or rapid plasma reagin. Forty-seven of the lots in the more recent study were reactive for hepatitis C antibody, but in the future we expect to see less hepatitis C reactivity in light of a new FDA requirement for donor screening and plasma processing. It is important then neither to overlook nor to overreact to hepatitis C antibody reactivity in a patient receiving IVIgG. Obviously, testing for hepatitis and cytomegalovirus (CMV) could also produce uninterpretable results.

Table 2. Partial List of Antibodies for Infectious Disease Markers Tested for and Identified in Various IVIgG Lots
No. lots positive
1986-1990
(n=165)
1991-1992
(n=51)
Hepatitis B surface165100%51100%
Hepatitis B core16097%2141%
Cytomegalovirus15896%51100%
Hepatitis A total165100%Not tested
HIV-16439%Not tested
HIV-1/2Not tested00%
Rapid plasma reagin4820%00%
Hepatitis CNot tested4792%

Another concern, especially in bone marrow transplant patients, is the presence of RBC alloantibodies (Tables 3 and 4). Since the recent introduction of IVIgG for CMV prophylaxis in bone marrow transplant recipients, a number of reports have described increased incidences of positive direct and indirect antiglobulin tests, confusing serologic results in pretransfusion testing, and rare cases of hemolysis (25-29). In a study by Robertson et al. of the serologic findings in 47 bone marrow transplant patients who received high doses of IVIgG (25), the frequency of positive direct antiglobulin results was 48.9% (P less than 0.001) versus 12.8% for a control group not receiving IVIgG. The frequency of positive indirect antiglobulin test results was 25.5% (P less than 0.001) in the IVIgG group versus 4.3% in the non-IVIgG group. The most frequently identified antibody specificities in the serum and the eluate were anti-A or anti-B, followed by anti-D and anti-K. In a test of 46 lots of IVIgG for antibodies against blood group antigens by Garcia et al. (26), anti-A or anti-B was detected in 88% of the lots, and indirect anti-human globulin was detected in 63% of the lots. Because IVIgG preparations, as we have pointed out, are manufactured from large donor plasma pools, they may contain varying amounts of isoagglutinins and other common alloantibodies such as anti-D and anti-K (5,30). The reported titers of isoagglutinins or anti-D in IVIgG preparations range from 0 to 128 (5,31,32) (Table 5).

Table 3. Isohemagglutinins in IVIgG Lots
No. lots positive
1986-1990
(n=165)
1991-1992
(n=49)*
Anti-A and anti-B11771%2959%
Anti-A2012%1327%
Anti-B85%48%
No isohemagglutinin2012%36%

*Two lots of 51 not tested.

Table 4. RBC Alloantibodies in IVIgG Lots
No. lots
1986-1990
(n=165)
1991-1992
(n=47)*
No evidence of RBC alloantibodies8451%817%
RBC alloantibodies present8149%3983%

*Four lots of 51 not tested.

Table 5. RBC Alloantibodies Tested for and Identified in IVIgG Lots, 1986-1990 and 1991-1992
No. lots positive
1986-1990
(n=81)
1991-1992
(n=39)
Anti-D3847%923%
Anti-K22%13%
Anti-CNo data13%
Anti-K, anti-D, and anti-LebNo data13%
Anti-K, anti-D, anti-Leb, and anti-CNo data13%
Anti-C and anti-DNo data13%
UnidentifiableNo data718%
Anti-K and unidentifiableNo data25%
Anti-D and anti-K22%615%
Anti-D and unidentifiable1215%821%
Anti-D, anti-K, and unidentifiableNo data1231%
With specific RBC alloantibodies4859%No data
Reactive with all cells tested2835%No data
Unidentifiable RBC alloantibodies1316%No data

In addition to IVIgG, other therapeutic immunosuppressive agents that have effects similar to those of IVIgG and are used in bone marrow transplant patients, such as anti-lymphocyte globulin and anti-thymocyte globulin, may create problems during compatibility testing. For instance, in a series of 37 patients studied by Swanson et al., 8 had positive direct antiglobulin tests and positive RBC antibody screens, 15 developed RBC antibodies detectable at room temperature, 4 developed RBC antibodies detectable at 37 degrees Celsisus in albumin, and 33 developed RBC antibodies detectable in the anti-human globulin phase (33). The compatibility problems were resolved by adsorbing the anti-human globulin with RBCs coated with anti-lymphocyte globulin, which allowed the substance in the anti-lymphocyte globulin reacting with the anti-human globulin to be removed.

Hemolytic episodes resulting from passively acquired IgG antibodies are another problem in IVIgG administration, although such episodes are usually mild and self-limiting. For instance, Robertson et al., in their study of 47 bone marrow transplant recipients after IVIgG therapy, observed no significant hemolysis associated with passive transfer of antibodies (25). On the other hand, a recent report by Copelan et al. described two patients who had clinically significant hemolysis after receiving large doses of IVIgG (27). One of these two patients had an acute episode of hemolysis after receiving two lots of IVIgG. Anti-A and anti-D were found in the patient's RBC eluate and anti-A and anti-D titers of 32 and 2, respectively, were demonstrated in the IVIgG lots. Although low titers of antibodies in IVIgG preparations have been assumed not to cause clinically significant hemolysis in patients (32), the two case reports by Copelan et al. illustrate rare exceptions (27).

Isohemagglutinins pose another problem. We and others have reported previously on the not unexpected presence of isohemagglutinins in IVIgG preparations (24,34). The presence of these antibodies, however, places a heavy burden on the logistics of the blood supply, especially in the case of IVIgG-dependent patients. If such patients are also transfusion dependent, then in most cases they must be given O, Rh- blood. In this regard then, it would be most helpful and logical for the manufacturers of IVIgG preparations to adsorb out these antibodies. However, although the technology to do this is available, such antibody removal is apparently not required by the FDA, according to the manufacturers' representatives.

It is interesting to note that in our 1991 study, roughly 50% of the IVIgG preparations showed no evidence of RBC alloantibodies (24). However, in our more recent review, only 17% of the IVIgG preparations were free of such antibodies. The reason for this is as yet unclear to us.

The list of RBC alloantibodies discussed here is not exhaustive and represents only the extent of our testing. Yet, all the alloantibodies we have discussed cloud the immunohematologic picture and further strain the process of ensuring a safe supply of blood components for transfusion-dependent patients (Tables 4-6).

Table 6. Impact of IVIgG on the Clinical Laboratory and the Transfusion Service
Patients on IVIgG may test false-positive in IgG-based assays.
Transfusion service is forced to use elaborate methods to identify RBC alloantibodies.
Transfusion-dependent patients on IVIgG deplete the O, Rh-negative blood inventory.

Our more recent analysis of IVIgG lots showed that other antibodies, such as anti-C, anti-c, and anti-Leb, also further complicate the workup and support of patients dependent simultaneously on IVIgG therapy and transfusions. To handle such a situation effectively, a hospital's transfusion service should be aware of patients receiving IVIgG. In hospitals in which the pharmacy dispenses IVIgG, this is not so easy information links must be established between the pharmacy and the transfusion service to provide the names of each patient receiving IVIgG, the IVIgG dosage, the lot number, and the date of infusion. However, in other settings in which the transfusion service itself dispenses IVIgG, it is very easy and convenient for the service to keep track of who gets it.

At our institution, empty IVIgG vials are saved by the pharmacy and delivered immediately to the transfusion service each time a patient receives an IVIgG infusion. Our transfusion service then records the lot number and date of infusion of IVIgG in the patient's file. The transfusion service also is usually able to retrieve enough IVIgG from the empty vials to screen the lots for the presence of RBC alloantibodies. Subsequently, when a request for transfusion for any patient on record is received, a rapid cross-check of the transfusion file by the transfusion service staff can alert them to potential serologic problems. Often, the antibody profiles detected in the lots of IVIgG resemble those found in the serum and eluate of patients receiving IVIgG. Therefore, the transfusion service must thoroughly investigate the blood serum antibody profiles of all transplant patients to determine what type of IVIgG solutions should be administered. These steps are valuable in resolving complex serologic problems.

Unavoidably, the clinical laboratory must establish methods and procedures to avoid false-positive pitfalls triggered by IVIgG infusions. On the other hand, the transfusion service must seek out information about patients receiving IVIgG therapy so that appropriate hemotherapeutic schemes can be provided in a timely manner. Consequently, the clinical laboratory and the transfusion service should both be in the information loop regarding patients receiving IVIgG. Furthermore, each IVIgG lot being used should be analyzed to determine what contaminating antibodies may be present and may produce spurious laboratory test results, results that may in turn trigger unwarranted clinical speculation and the ordering of additional but unnecessary therapeutic and diagnostic procedures.

In dealing with IVIgG, the clinical laboratory, pharmacy, transfusion service, attending physician, and nursing staff must all have open channels of communication and information to prevent unnecessary testing and workups in such patients. As stated above, those clinical and transfusion professionals who deal with IgA-deficient patients must be particularly alert when using IVIgG preparations since such preparations may contain various amounts of IgA and since the infusion of IVIgG into an IgA-deficient patient with IgA antibodies can unleash a severe anaphylactic reaction.

Another note of caution relates to allergic transfusion reactions that may ensue during or following a posttransfusion IVIgG infusion. It may happen that a nursing staff will overlook the infusion of IVIgG in such cases and report only that the patient in question received some type of transfusion. Although the transfusion reaction should not be overlooked, knowing that IVIgG was infused would clearly help in understanding the allergic reaction.

Without a doubt, IVIgG therapy is a constantly expanding modality. Patients who receive it do experience beneficial effects, and the list of disease entities in which IVIgG can be tried and administered is currently limitless. In view of this, the clinical laboratory and transfusion service must develop a proactive strategy for dealing with the testing interferences induced by IVIgG infusions.

  1. Stiehm ER, Ashida E, Kim KS, et al. Intravenous immunoglobulins as therapeutic agents. Ann Intern Med 207:367-382, 1987.
  2. Buckly RH, Schiff RI. The use of intravenous immunoglobulin in immunodeficiency diseases. N Engl J Med 325:110-117, 1991.
  3. Newland AC. Clinical use of intravenous immunoglobulin in blood disorders. Blood Rev 2:157-167, 1988.
  4. Gardi A. Quality control in the production of an immunoglobulin for intravenous use. Blut 48:337-344, 1984.
  5. Romer J, Morgenthaler JJ, Scherz R, et al. Characterization of various immunoglobulin preparations for intravenous application: I. Protein composition and antibody content. Vox Sang 42:62-73, 1982.
  6. Romer J, Spath PJ, Skvaril F, et al. Characterization of various immunoglobulin preparations for intravenous application: II. Complement activation and binding to Staphylococcus protein A. Vox Sang 42:74-80, 1982.
  7. Romer J, Spath PJ. Molecular composition of immunoglobulin preparations and its relation to complement activation. In Nydegger UE (ed), Immunohemotherapy: A Guide to Immunoglobulin Prophylaxis and Therapy. London, Academic Press, 1981, p. 123.
  8. Skvaril F, Roth-Wicky B, Barandum S. IgG subclasses in human-gamma-globulin preparations for intravenous use and their reactivity with Staphylococcus protein A. Vox Sang 38:147, 1980.
  9. Skvaril F. Qualitative and quantitative aspects of IgG subclasses in i.v. immunoglobulin preparations. In Nydegger UE (ed), Immunohemotherapy: A Guide to Immunoglobulin Prophylaxis and Therapy. London, Academic Press, 1981, p. 113.
  10. Skvaril F, Barandun S. In vitro characterization of immunoglobulins for intravenous use. In Alving BM, Finlayson JS (eds), Immunoglobulins: Characteristics and Uses of Intravenous Preparations, DHHS Publication No. (FDA)-80-9005. U.S. Government Printing Office, 1980, pp. 201-206.
  11. Burckhardt JJ, Gardi A, Oxelius V, et al. Immunoglobulin G subclass distribution in three human intravenous immunoglobulin preparations. Vox Sang 57:10-14, 1989.
  12. Morell A, Skvaril F. Stuktur und biologische eigenschaften von immunoglobulinen und gamma-globulin-praparaten: II. Eigenschaften von gamma-globulin-praparaten. Schweiz Med Wochenschr 110:80, 1980.
  13. Morell A, Schurch B, Ryser D, et al. In vivo behavior of gamma globulin preparations. Vox Sang 38:272, 1980.
  14. Imbach P, Barandun S, d'Apuzzo V, et al. High-dose intravenous gamma globulin for idiopathic thrombocytopenic purpura in childhood. Lancet 1:1228, 1981.
  15. Barandun S, Morell A, Skvaril F. Clinical experiences with immunoglobulin for intravenous use. In Alving BM, Finlayson JS (eds), Immunoglobulins: Characteristics and Uses of Intravenous Preparations. DHHS Publication No. (FDA)-80-9005. U.S. Government Printing Office, 1980, pp. 31-35.
  16. Barandun S, Morell A. Adverse reactions to immunoglobulin preparations. In Nydegger UE (ed), Immunohemotherapy: A Guide to Immunoglobulin Prophylaxis and Therapy. London, Academic Press, 1981, p. 223.
  17. Heiner DC. Significance of immunoglobulin G subclasses. Am J Med 76:1-5, 1984.
  18. Skvaril F. Clinical relevance of IgG subclasses. In Morell A, Nydegger UE (eds), Clinical Use of Intravenous Immunoglobulins. London, Academic Press, 1986, pp. 37-45.
  19. Ochs HD, Wedgwood RJ. IgG subclass deficiencies. Annu Rev Med 38:325-340, 1987.
  20. Schur PH. IgG subclasses: A review. Ann Allergy 58:89-99, 1987.
  21. Berger M. Immunoglobulin G subclass determination in diagnosis and management of antibody deficiency syndromes. J Pediatr 110:325-328, 1987.
  22. Skvaril F, Gardi A. Differences among available immunoglobulin preparations for intravenous use. Pediatr Infect Dis J 7:S43-S48, 1988.
  23. Copelan EA, Avalos BR, Kapoor N, et al. Alternate applications of immunoglobulin following bone marrow transplantation. Semin Hematol 29:96-99, 1992.
  24. Lichtiger B, Rogge K. Spurious serologic test results in patients receiving infusions of intravenous immune gammaglobulin. Arch Pathol Lab Med 115:467-469, 1991.
  25. Robertson VM, Dickson IG, Romond EH. Positive antiglobulin test due to intravenous immunoglobulin in patients who received bone marrow transplants. Transfusion 27:28-31, 1987.
  26. Garcia L, Huh YO, Fischer HE. Positive immunohematologic and serologic test results due to high dose intravenous immunoglobulin administration (letter). Transfusion 27:503, 1987.
  27. Copelan EA, Strohm PL, Kennedy MS. Hemolysis following intravenous immune globulin therapy. Transfusion 26:410-412, 1986.
  28. Steiner EA, Butch SH, Carey JL, et al. Passive anti-D from intravenous immune serum globulin (letter). Transfusion 23:363, 1983.
  29. Whitsett CF, Pierce JA, Daffin LE. Positive direct antiglobulin tests associated with intravenous gamma globulin use in bone marrow transplant recipients. Transplantation 41:663-664, 1986.
  30. Good RA (ed). Intravenous immune globulin and the compromised host: Proceedings of a symposium. Am J Med 716(3A), 1984.
  31. Gordon JM, Cohen P, Finlayson JS. Levels of anti-A and anti-B in commercial immune globulin. Transfusion 20:90-92, 1980.
  32. Hoppe I. Antibody screening of commercial immunoglobulin preparations, erythrocytes, HLA and autoantibodies. Blut 39:9, 1979.
  33. Swanson JL, Issitt CH, Mann EW, et al. Resolution of red cell compatibility testing problems in patients receiving anti-lymphoblast or anti-thymocyte globulin. Transfusion 24:141-147, 1984.
  34. Lang GE, Veldhuis B. Immune serum globulin--a cause for anti-Rh(D) passive sensitization. Am J Clin Pathol 60:205-207, 1973.

CURRENT ISSUES IN TRANSFUSION MEDICINE
Volume 3, Number 2
Copyright 1995 The University of Texas M. D. Anderson Cancer Center, Houston, Texas


HISTORY

Wassermann test or Wassermann reaction

It was the First blood test for syphilis and the first test in the nontreponemal tests (NTT) category developed by Wassermann, Julius Citron, and Albert Neisser in 1906

It is based on Complement fixation principle in which a sample of blood or CSF was taken and introduced to the antigen which was cardiolipin extracted from bovine muscle or heart. Treponemal nonspecific antibodies react with the lipid – the Wassermann reaction (WR) of antiphospholipid antibodies (APAs)

The intensity of the reaction (classed 1, 2, 3, or 4) indicates the severity of the condition

Newer NTTs, such as the rapid plasma reagin (RPR) and VDRL tests, have mostly replaced it.


Executive Summary and Background

Executive Summary

Serologic tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are now widely available. Unlike nucleic acid amplification tests (NAAT) which detect viral RNA, antibody-based assays measure the host&rsquos humoral immune response to current or past infection. Anti-SARS-CoV-2 antibodies typically become detectable more than two weeks after the onset of symptoms (Figure 1). As a result, SARS-CoV-2 serology lacks sufficient sensitivity to confidently exclude the diagnosis of coronavirus disease 2019 (COVID-19) when antibodies are not detected in the acute phase of illness. NAAT remains the diagnostic modality of choice for acute infection. Antibody testing, however, may be useful as an adjunct to NAAT at later time points following infection. In general, IgM tests tend to have lower sensitivity to detect past infection than IgG or total antibody tests. Assays designed to detect and differentiate IgM and IgG in combination, where the detection of either IgM or IgG is used to define a positive test result, and IgA tests tend to have lower specificity to detect past infection compared to IgG only or total antibody tests. Test specificity is especially important for large serosurveillance studies when the prevalence of prior infection in the community is expected to be low. To be of value, anti-SARS-CoV-2 antibody tests are required to have high clinical sensitivity and specificity (i.e., > 99.5%).

In addition to use in epidemiologic studies, the panel identified two clinical scenarios where antibody testing was felt to have potential utility for diagnosis. Serologic testing may be helpful in the evaluation of individual patients with a high clinical suspicion for COVID-19 when the results of molecular diagnostic testing are repeatedly negative or such testing was not performed. The sensitivity and specificity of IgG and total antibody is optimal three to four weeks after the onset of symptoms. At the current time, little data exists in the fifth week post-symptom onset to judge serologic test performance at later periods after infection. Detection of anti-SARS-CoV-2 antibodies is also useful for assessments of suspected multisystem inflammatory syndrome in children. For symptomatic patients, optimal serology result interpretation requires careful determination of the timing of testing relative to symptom onset combined with assessments of disease severity. Based on the available evidence at this time, serologic tests should not be used to determine immunity or risk of re-infection. Thus, anti-SARS-CoV-2 antibody detection cannot inform decisions to discontinue physical distancing or lessen the use of personal protective equipment.

Summarized below are specific recommendations and comments related to the use of SARS-CoV-2 serologic testing in clinical practice and public health. A detailed description of background, methods, evidence summary and rationales that support each recommendation can be found online in the full text.

Recommendations

  • Recommendation 1:The IDSA panel suggests against using serologic testing to diagnose SARS-CoV-2 infection during the first two weeks (14 days) following symptom onset (conditional recommendation, very low certainty of evidence).
  • Recommendation 2:When SARS-CoV-2 infection requires laboratory confirmation for clinical or epidemiological purposes, the IDSA panelsuggests testing for SARS-CoV-2 IgG or total antibody three to four weeks after symptom onset to detect evidence of past SARS-CoV-2 infection (conditional recommendation, very low certainty of evidence).
    • Remark &ndash When serology is being considered as an adjunct to NAAT for diagnosis, testing three to four weeks post-symptom onset maximizes the sensitivity and specificity to detect past infection.
    • Remark &ndash Serosurveillance studies should use assays with high specificity (i.e., > 99.5%), especially when the prevalence of SARS-CoV-2 in the community is expected to be low.
    • Remark &ndash IgM or IgG combination tests are those where detecting either antibody class is used to define a positive result.
    • Remark &ndash When serology is being considered as an adjunct to NAAT for diagnosis, testing three to four weeks post-symptom onset maximizes the sensitivity and specificity to detect past infection.

    Background

    Since its emergence in December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused over 21 million known infections and nearly 770,000 deaths worldwide [1]. Definitive diagnosis of coronavirus disease 19 (COVID-19), the illness caused by SARS-CoV-2 infection, relies on the direct detection of virus-specific RNA or virus-specific glycoprotein antigens in respiratory tract specimens. Serologic tests that detect the host antibody response to SARS-CoV-2 may also help to confirm the presence of current or past infection using blood samples.

    Coronavirus genomes encode four major structural proteins including spike (S), envelope (E), membrane (M) and nucleocapsid (N). Both the S and N proteins of SARS-CoV-2 have been shown to be immunogenic in humans and current serologic tests target antibodies directed against these antigens [2]. The S protein is the most exposed viral protein and is responsible for viral attachment and entry into the host cell via binding to the angiotensin-converting enzyme 2 (ACE-2) receptor [3]. The S protein is comprised of an N-terminal S1 subunit, involved in virus-receptor binding, and a C-terminal S2 subunit that is involved in fusion to the host cell membrane. The S1 subunit is further divided into the N terminal domain (NTD) and a receptor binding domain (RBD). There has been particular focus on the SARS-CoV-2 RBD for vaccine development and targeted antibody therapies because neutralizing antibodies against this region effectively block viral entry [4, 5]. The N protein is an RNA-binding protein that is abundantly expressed during infection and plays an important role in RNA transcription and replication [6].

    There are two general types of antibodies, neutralizing antibodies (nAbs) and non-neutralizing (also known as binding antibodies) [7]. Neutralization is defined as the loss of infectivity that occurs when a nAb binds to a viral particle. Virus-specific or vaccine-induced nAbs can play a crucial role in controlling viral infection, but definitive data is lacking to know whether individuals with detectable anti-SARS-CoV-2 nAbs are protected against reinfection. In comparison, binding antibodies are characterized by their inability to prevent viral infection of permissive cells. Regardless of their function, both types of virus-specific antibodies are potentially useful as diagnostic indicators of current or past infection.

    Commercially available anti-SARS-CoV-2 antibody tests use different technologies to qualitatively measure single immunoglobulin classes (IgM, IgG or IgA) or total antibody, but do not differentiate nAbs from binding antibodies. IgM antibodies directed against microorganisms are typically produced first after infection and are used as a measure of recent infection. IgG antibodies generally develop later after IgM and remains elevated for months to years after infection. Although IgM antibodies can be detected within the first two weeks of symptoms in some patients, SARS-CoV-2 infection appears unusual in that IgM and IgG more commonly rise together, more than two weeks after the onset of symptoms [8]. Secretory IgA is important for mucosal immunity. IgA can also be detected systemically in certain types of infection including SARS-CoV-2, but comparatively little is known about the kinetics of IgA in blood. The components of &ldquototal antibody&rdquo presumably include IgM and IgG and theoretically other antigen-specific immunoglobulins as well.

    Given that the majority of the population has previously been exposed to seasonal human coronaviruses (HCoV), and these viruses may share similar structure with SARS-CoV-2, an essential part of serologic test development and validation is to assure that the anti-SARS-CoV-2 antibodies detected by a given assay do not cross react with other coronaviruses (e.g., HCoV-229E, HCoV-NL63, HCoV-OC43 or HCoV-HKU1). Specificity studies typically involve analyzing archived sera obtained before the identification of COVID-19 as a clinical entity, as well as assessing for potential interfering substances such as auto-antibodies or heterophile antibodies.

    The most common clinical diagnostic platforms utilized for SARS-CoV-2 include lateral flow (LF) devices, enzyme linked immunosorbent assays (ELISA) and chemiluminescent immunoassays (CIA). Lateral flow assays typically require a drop of blood (or serum or plasma) applied to a test strip, with results read in approximately 15-30 minutes. These devices are suitable for point-of-care testing and have potential to be deployed in the field as a part of large serological surveys. ELISA comes in a variety of different formats. Typically, a bound antigen-antibody complex is detected using a type-specific secondary antibody linked to a substrate that generates a colorimetric or fluorescent signal. CIA methods are similar to ELISA but use chemical probes that emit light instead of enzymatic substrates. Both ELISA and CIA are clinical laboratory-based methods amendable to high throughput testing using serum, plasma or potentially dried blood spots. At this time, neutralization assays are mainly used in research settings or offered as laboratory developed tests by reference laboratories.

    In the United States, the Food and Drug Administration (FDA) currently requires Emergency Use Authorization (EUA) to market a SARS-CoV-2 antibody test. This means that commercial manufacturers and clinical laboratories with laboratory-developed tests must submit performance data to the FDA for review. Early in the pandemic, however, official EUA review was voluntary. Test developers were only expected to internally validate their tests and notify the FDA of their intent to market. As a result, the market was flooded with poorly performing assays. In response the FDA subsequently issued a &ldquoremoved&rdquo test list that includes tests where significant performance problems were identified, assays for which official EUA review was not appropriately submitted or assays voluntarily withdrawn by the developer.

    Many different serologic tests for SARS-CoV-2 have become commercially available in a short amount of time. The incredible speed of development has significantly outpaced rigorous assessments of test performance. Therefore, IDSA convened an expert panel to systematically review the available serologic literature, compare pooled estimates of test accuracy and make evidence-based recommendations for informed use in clinical practice.


    Conclusions

    The rapid development and implementation of a range of diagnostic assays is undoubtedly an essential part of the coordinated response to a new pathogen. However, the limitations of novel assays and of clinicians' understanding of these must be considered. 4,5 To our knowledge, this is the first study to investigate clinicians' interpretive response to novel SARS-CoV-2 serology. There are significant limitations to our study design, both in our modest number of survey responses and the necessity for rapid design and implementation due to the evolving nature of the pandemic. As free text comments were optional, analysis of these is also limited. However, we highlight that there is likely to be marked variation in the clinical interpretation of SARS-CoV-2 serology results as they become available. Further research in this area is urgently warranted, as this may have serious implications for ongoing public health efforts to maintain social distancing measures and the isolation of patients affected by COVID-19. Proactive interpretive support, which includes ‘narrative comments’ from laboratory and infectious diseases specialists, is strongly recommended (Box 1).

    Examples of interpretative comments that may be useful in reporting SARS-CoV-2 serology


    Development and Validation of a Serologic Test Panel for Detection of Powassan Virus Infection in U.S. Patients Residing in Regions Where Lyme Disease Is Endemic

    FIG 1 Titration of acute-phase tick-borne disease (TBD) samples in indirect immunofluorescence assay (IFA) to determine optimal screening dilutions. (a) Serial 2-fold dilutions of acute-phase TBD sample with Powassan virus (POWV) plaque reduction neutralization test (PRNT) titer of 1:320 to determine optimal screening dilution for IgM IFA. (b) Serial 2-fold dilutions of acute-phase TBD sample with POWV PRNT titer of 1:160 to determine optimal screening dilution for IgG IFA.
    Sample
    no.
    Result for assay:
    TBE-C EIA
    IgM
    POWV IFA
    IgM
    TBE-C EIA
    IgG
    POWV IFA
    IgG
    POWV PRNT90
    titer
    1+ND b ND≥1:401:5,120
    2+ND+≥1:401:640
    3+ND+≥1:401:2,560
    4ND≥1:20+≥1:401:320
    5+≥1:20ND≥1:401:5,120
    6ND≥1:20+≥1:1001:10,240
    7+ND+≥1:1001:40,960
    8NDNDNDND1:20
    9+≥1:20ND≥1:401:20

    Analytical specificity.

    FIG 2 Yellow fever virus (YFV) vaccine recipient plasma samples in Powassan virus (POWV) indirect immunofluorescence assay (IFA) to determine optimal screening dilutions to eliminate cross-reactivity. (Top) YFV IgG-positive sample 7 years postvaccine assayed at 1:20 (left) and 1:40 (right) dilutions in IgG IFA. (Bottom) YFV IgM-positive sample 4 weeks postvaccine assayed at 1:10 (left) and 1:20 (right) dilutions in POWV IgM IFA.

    Serological test

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    Serological test, also called serology test or antibody test, any of several laboratory procedures carried out on a sample of blood serum (the clear liquid that separates from the blood when it is allowed to clot) for the purpose of detecting antibodies or antibody-like substances that appear specifically in association with certain diseases. There are different types of serological tests—for example, flocculation tests, neutralization tests, hemagglutinin-inhibition tests, enzyme-linked immunosorbent assays (ELISAs), and chemiluminescence immunoassays.

    Among flocculation tests, complement-fixation tests are the most common. These are based on the precipitation, or flocculation, that takes place when an antibody and specially prepared antigens (substances that provoke antibody production in the body) are mixed together. Neutralization tests depend on the capacity of an antibody to neutralize the infectious properties of the infectious organisms. Hemagglutinin-inhibition tests are based on the ability of viruses to cause the red blood cells of certain animal species to agglutinate (congeal, or clump together) this agglutination will be prevented by the antibody. ELISAs make use of fluorescent, light (chemiluminescent), or colorimetric signal detection the signals are produced by enzymatic reactions that occur during the detection and quantification of a specific antigen or antibody in a solution. Chemiluminescence immunoassays are based on the detection of light signals emitted through chemical reactions between enzymes or chemical probes that bind to antibodies.

    Serological testing is particularly helpful in the diagnosis of certain bacterial, parasitic, and viral diseases, including Rocky Mountain spotted fever, influenza, measles, polio, yellow fever, and infectious mononucleosis. It is also useful in the detection of autoantibodies (harmful antibodies that attack components of the body) that are involved in autoimmune diseases, such as rheumatoid arthritis. As a practical mass-screening tool, serological testing has proved valuable in the detection of diseases such as syphilis, HIV/AIDS, and epidemic and pandemic infectious diseases (e.g., influenza and coronavirus disease). See also blood analysis.

    The Editors of Encyclopaedia Britannica This article was most recently revised and updated by Kara Rogers, Senior Editor.


    Background

    Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) initiates a humoral immune response that produces antibodies against specific viral antigens such as the nucleocapsid (N) protein and spike (S) protein, which include specific anti-S protein antibodies that target the spike&rsquos S1 protein subunit and receptor binding domains (RBD). Serologic tests can detect the presence of these antibodies in serum within days to weeks following acute infection. However, serologic testing should not be used to diagnose acute SARS-CoV-2 infection. Serologic tests can identify persons with resolving or past SARS-CoV-2 infection and thereby help scientists and public health experts better understand the epidemiology of SARS-CoV-2 individuals and populations at higher risk of infection. Although the immune correlates of protection are not fully understood, evidence indicates that antibody development following infection likely confers some degree of immunity from subsequent infection for at least 6 months. However, it is not known to what extent emerging viral variants may impact immunity from subsequent infection.


    Roadblocks to Success

    Accuracy concerns and questions about authorization guidelines have generated hesitancy amongst clinical laboratories performing serology tests. Much of this uncertainty is based on limited data. Ironically, the direct applications of serology testing create inherent roadblocks to answering these questions. When laboratories are making real-time decisions about the allocation of resources, time and expertise, the fact that serology testing is not intended for diagnostic use is impactful. Hospital labs must prioritize the identification of acute cases of illness in order to contain the spread of disease and provide effective treatment for critically ill patients. In short, surveillance studies are useful for epidemiologic purposes, but not the first priority in triage situations. That&rsquos why serology tests are being predominately conducted in large clinical labs and research facilities at this time.

    It&rsquos therefore valuable to conclude with a summary of the current recommended uses for serology testing.

    When COVID-19 Serology Testing Should Be Used

    • The primary application of serology testing is the identification of individuals who have previously been infected with SARS-CoV-2. This knowledge can be used to guide epidemiology and seroprevalence studies, as well as facilitate contact tracing.
    • Serology tests may also be used to identify potential convalescent plasma donors and to evaluate the immune response to candidate vaccines.
    • Finally, there is potential for serology tests to aid in the diagnosis of COVID-19 in RT-PCR negative patients who present later during disease course.

    When COVID-19 Serology Testing Should NOT Be Used

    • Serology testing should not be used to diagnose acute or recent cases of COVID-19.
    • At this time, serology tests cannot be used to definitively determine if a patient has developed protective immunity.
    • Because of the above limitations, SARS-CoV-2 serology testing should not be used to guide personal protective equipment (PPE) use or adherence to social distancing practices.

    &ldquoIn absence of approved and proven therapies, diagnostics become particularly important,&rdquo said Dr. Bertuzzi. &ldquo70% of doctors&rsquo decisions are based on tests performed in the lab.&rdquo But we need to continue to be cautious.

    &ldquoWe need to be clear about what testing is for, and we need to do the careful science to not unnecessarily waste the resources we have developed,&rdquo added Dr. Gerberding. &ldquoWe&rsquore nowhere near herd immunity, so we need to find other solutions to restart our economy.&rdquo The American Society for Microbiology has developed step-by-step procedures to help labs develop efficient and effective verification protocols for COVID-19 serologic assays.


    Lab 17: Serology, Direct and Indirect Serologic Testing - Biology

    We prospectively examined the effectiveness of diagnostic tests for anaplasmosis using patients with suspected diagnoses in France. PCR (sensitivity 0.74, specificity 1) was the best-suited test. Serology had a lower specificity but higher sensitivity when testing acute and convalescent samples. PCR and serology should be used in combination for anaplasmosis diagnosis.

    Human granulocytic anaplasmosis (HGA) is a tickborne intracellular bacterial infection caused by Anaplasma phagocytophilum. The disease is present in North America, Europe, and northern Asia, areas with Ixodes ricinus ticks, the primary vector for transmission to humans (1,2). Clinical manifestations of disease include acute fever, headache, and myalgia occurring 2–3 weeks after tick bite. Diagnosis requires the isolation of A. phagocytophilum in blood culture, the presence of morulae in polymorphonuclear cells after May Grünwald-Giemsa staining of peripheral blood smears, positive serologic results (seroconversion or high titer of specific antibodies), or a positive A. phagocytophilum PCR result. The May Grünwald-Giemsa stain test has a low sensitivity (3) PCR and serology are more widely available, but their diagnostic value is not well established. The aim of our study was to compare the diagnostic values of the available microbiological tests in a prospectively selected series of patients with clinical signs and symptoms consistent with an HGA diagnosis.

    The Study

    In this prospective, multicenter study, we enrolled symptomatic patients living in Alsace, a region of northeastern France where tickborne diseases are highly endemic. Patients gave written, informed consent to participate in our study, which was approved by the ethics committee of the University Hospital of Strasbourg (Strasbourg, France).

    We included patients if they had 1 of the following combinations of signs and symptoms occurring no more than 4 weeks after a tick bite: 1) fever or other symptom presumed related to a tick bite, 2) fever plus thrombocytopenia with or without leukopenia or elevated liver enzyme levels, 3) thrombocytopenia with or without leukopenia, or 4) elevated liver enzyme levels without fever. The first visit included clinical and epidemiologic evaluations and the collection of blood samples for A. phagocytophilum serology, May Grünwald-Giemsa staining, and A. phagocytophilum–specific PCR. We did not culture for A. phagocytophilum. An etiologic investigation was also conducted to obtain a differential diagnosis. After > 4 weeks, a second visit was scheduled to obtain a clinical evaluation, A. phagocytophilum serology, and (if necessary) a complete differential diagnosis.

    We stratified patients into 3 groups on the basis of their diagnosis. One group included controls, who were patients with a clinical and microbiologically confirmed nonanaplasmosis diagnosis. The second group included anaplasmosis patients defined by > 1 of the following criteria: intraleukocyte morulae on blood smears, a positive PCR result for Anaplasma, a 4-fold increased antibody titer for A. phagocytophilum in the follow-up sample or a seroconversion (i.e., change in antibody titer from negative in first sample to > 1:64 in second sample), or a high antibody titer for Anaplasma ( > 1:256) by indirect immunofluorescence antibody assay. The third group were patients without any diagnosis.

    We performed DNA extraction, PCR, and serologic testing blinded to sample identification as previously described (4). The PCR targeted the A. phagocytophilum msp2/p44 gene. We performed serologic testing using the Anaplasma phagocytophilum IFA IgG assay (Focus Diagnostics, http://www.focusdx.com) (4). Trained staff examined May Grünwald-Giemsa–stained smear preparations of whole blood samples for intracellular morulae. We collected data by using EpiData version 3.1.2701.2008 (http://epidata.dk) and extracted data to Excel spreadsheets (Microsoft, https://www.microsoft.com) for analysis. After patient stratification, we estimated the sensitivity and specificity of the different diagnostic tests.

    Figure. Distribution of positive diagnostic test results for patients with confirmed human granulocytic anaplasmosis, France, May 2010–July 2012.

    During May 2010–July 2012, we enrolled 155 patients into the study, 25 of whom did not complete the second visit. None of these 25 patients had a positive PCR result or an antibody titer > 1:256 at the first visit. The remaining 130 patients completed both study visits and were thus included in the study evaluation. Of these 130 patients, 19 had confirmed anaplasmosis diagnoses and 36 were controls with confirmed nonanaplasmosis diagnoses (infections with Borrelia burgdorferi, Epstein-Barr virus, cytomegalovirus, HIV, tick-borne encephalitis virus, Leptospira spp., Babesia spp., parvovirus B19, hantavirus, Francisella tularensis, Plasmodium spp., and Aeromonas spp.). Of the patients with HGA, 84.2% (16/19) met the serologic criteria and 73.7% (14/19) met the PCR criteria (Table Figure). Fever, the most frequent symptom (89%), was associated with joint and muscle pain. Cytopenia of platelets, neutrophils, or both (74%) and elevated liver enzyme levels (63%) were frequently present.

    Calculations of the diagnostic value of each test method showed that PCR had a sensitivity of 0.74 and a specificity of 1 and blood smear staining had a sensitivity of 0.21 and a specificity of 1. Seroconversion or a 4-fold increase of antibody titer had a sensitivity of 0.32 and specificity of 0.97, an antibody titer > 1:256 had a sensitivity of 0.58 and specificity of 0.97, and overall serology had a sensitivity of 0.84 and specificity of 0.97.

    The interval between the first and second serologic tests for most patients in the anaplasmosis group was 4–8 weeks (mean 49.8 days). Five patients had the second test >8 weeks after the first. Of these patients, 2 seroconverted, 1 experienced a substantial decrease in antibody titer, 1 experienced a substantial increase at week 12, and 1 had a stable antibody titer.

    Our study confirms PCR as the gold standard for diagnosis of HGA this test enabled rapid diagnosis during the acute stage of infection with good sensitivity and excellent specificity. However, the absence of a gold standard diagnostic test to compare our results with is a limitation to our study. A. phagocytophilum culture is the reference test for HGA diagnosis (5,6) but is not well suited for routine use because culturing is time-consuming and not widely performed. The diagnosis of anaplasmosis often involves assessing for the presence of morulae, but this test has low sensitivity (3). In our study, this test was of limited value for HGA diagnosis because whenever morulae were detected on blood smears > 1 of the other diagnostic tests were positive. However, May Grünwald-Giemsa staining is the quickest test to do, and when performed by trained staff, positive results are helpful for physicians.

    In clinical practice, diagnosis of HGA often relies on serology (79), but 2 limitations are associated with this method: a risk for false-negative results during the acute stage of infection because A. phagocytophilum antibodies are detected on average 11.5 days after symptom onset and a risk for false-positive results because Anaplasma antibodies are detectable in 86.4% of patients for 6–10 months and in 40% of patients up to 2 years after the initial infection (10). Positive serologic criteria are seroconversion, a 4-fold increase in antibody titer, or a stable and high antibody titer (11,12). In our study, we observed that each of these criteria can lead to misdiagnosis at the beginning of infection, as previously reported (13).

    PCR is considered the most effective diagnostic method during early stage A. phagocytophilum infection (14,15). Our results confirm this belief, despite our limitation of a small study population. However, if PCR is use alone, HGA might be underdiagnosed.

    Conclusions

    The presentation of fever in a patient with a history of tick bite does not qualify for an anaplasmosis diagnosis microbiological tests need to be performed. For anaplasmosis, PCR testing appears to be the most effective diagnostic tool. However, the sensitivity of PCR is <100%, and combining PCR with serologic testing at the first visit appears to be the best strategy for early diagnosis of acute anaplasmosis. In cases of high suspicion for HGA in patients without any diagnosis at the first visit, a second serologic test > 4 weeks later can be helpful. A multiplex approach could also be used in such cases to look for differential diagnoses.

    Dr. Hansmann is head of the Infectious Disease Department at Strasbourg University Hospital, Strasbourg, France a member of the Borreliosis group of the European Society of Clinical Microbiology and Infectious Diseases and involved in designing the tickborne disease national diagnosis and health plan for France. His research interests are tickborne diseases and diagnosis.

    Acknowledgment

    This study was supported by the French Hospital Clinical Research Program (PHRC HUS 2007–3960).


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