Immunodiagnostic Studies

Immunodiagnostic Studies


Immunodiagnostic or serodiagnostic testing studies antigen-antibody reactions for diagnosis of infectious disease, autoimmune disorders, immune allergies, and neoplastic disease. These modalities also test for blood groups and types, tissue and graft transplant matching, and cellular immunology. Blood serum is tested for antibodies against particular antigens—hence the term blood serology testing.

Antigens are substances that stimulate and subsequently react with the products of an immune response. They may be enzymes, toxins, microorganisms (e.g., bacterial, viral, parasitic, fungal), tumors, or autoimmune factors. Antibodies are proteins produced by the body’s immune system in response to an antigen or antigens. The antigen-antibody response is the body’s natural defense against invading organisms. Red blood cell groups contain almost 400 antigens. Immune reactions to these antigens result in a wide variety of clinical disorders, which can be tested (e.g., Coombs’ test).

Pathologically, autoimmune disorders are produced by autoantibodies—that is, antibodies against self. Examples include systemic rheumatic diseases, such as rheumatoid arthritis and lupus erythematosus.

Immunodeficiency diseases exhibit a lack of one or more basic components of the immune system, which includes B lymphocytes, T lymphocytes, phagocytic cells, and the complement system. These diseases are classified as primary (e.g., congenital, DiGeorge syndrome) and secondary (e.g., acquired immunodeficiency syndrome [AIDS]).

Hypersensitivity reactions are documented using immediate hypersensitivity tests and are defined as abnormally increased immune responses to some allergens (e.g., allergic reaction to bee stings or pollens). Delayed hypersensitivity skin tests are commonly used to evaluate cellmediated immunity. Histocompatibility antigens (transplantation antigens) and tests for human leukocyte antigen (HLA) are important diagnostic tools to detect and prevent immune rejection in transplantation.

Types of Tests

Many methods of varying sophistication are used for immunodiagnostic studies (Table 8.1).

Collection of Serum for Immunologic Tests

Specific antibodies can be detected in serum and other body fluids (e.g., synovial fluid, CSF).

1. Procure samples. For diagnosis of infectious disease, a blood sample (serum preferred) using a 7-mL red-topped tube should be obtained at illness onset (acute phase), and the other sample should be drawn 3 to 4 weeks later (convalescent phase). In general, serologic test usefulness depends on a titer increase in the time interval between the acute and the convalescent phase. For some serologic tests, one serum sample may be adequate if the antibody presence indicates an abnormal condition or the antibody titer is unusually high. See Appendix A for standard precautions.

2. Perform the serologic test before doing skin testing. Skin testing often induces antibody production and could interfere with serologic test results.

3. Label the sample properly and submit requested information. Place specimen in biohazard bag. Send samples to the laboratory promptly. Hemolyzed samples cannot yield accurate results. Hemoglobin in the serum sample can interfere with complement-fixing antibody values.

Interpreting Results of Immunologic Tests

The following factors affect test results:

1. History of previous infection by the same organism

2. Previous vaccination (determine time frame)

3. Anamnestic reactions caused by heterologous antigens: An anamnestic reaction is the appearance of antibodies in the blood after administration of an antigen to which the patient has previously developed a primary immune response.

4. Cross-reactivity: Antibodies produced by one species of an organism can react with an entirely different species (e.g., Tularemia antibodies may agglutinate Brucella and vice versa, rickettsial infections may produce antibodies reactive with Proteus OX19).

5. Presence of other serious illness states (e.g., lack of immunologic response in agammaglobulinemia, cancer treatment with immunosuppressant drugs)

6. Seroconversion: the detection of specific antibody in the serum of an individual when this antibody was previously undetectable

TABLE 8.1 Some Tests That Determine Antigen-Antibody Reactions

Name of Test

Observable Reaction

Visible Change

Tests for

Agglutination, hemagglutination (HA), immune hemagglutination assay (IHA)

Particulate antigen reacts with corresponding antibody; antigen may be in form of RBCs (hemagglutination, latex, or charcoal coated with antigen).


Treponemal, heterophile, and cold agglutinin antibodies

Precipitation (e.g., immunodiffusion [ID], counterimmunoelectrophoresis [CIE])

Soluble antigen reacts with corresponding antibody by ID or count.


Fungal antibodies, food poisoning

Complement fixation (CF)

Competition between two antigen-antibody systems (test and indicator systems)

Complement activation, hemolysis

Viral antibodies

Immunofluorescence (e.g., indirect fluorescent antibody [IFA])

Fluorescenttagged antibody reacts with antigen-antibody complex in the presence of ultraviolet light.

Visible microscopic fluorescence

Antinuclear antibodies (ANAs); antimitochondrial antibodies (AMAs)

Enzyme immunoassay (EIA)

Enzymes are used to label induced antigen-antibody reactions.

Chromogenic fluorescent or luminescent change in substrate

Hepatitis and human immunodeficiency virus (HIV) (screening)

Enzyme-linked immunosorbent assay (ELISA)

Indirect EIA for quantification of an antigen or antibody enzyme and substrate

Color change indicates enzyme substrate reaction.

Lyme disease, Epstein-Barr virus, extractable nuclear antibodies (connective tissue/systemic rheumatic disease)

Immunoblot (e.g., Western blot [WB])

Electrophoresis separation of antigen subspecies

Detection of antibodies of specific mobility

Confirms HIV-1

Polymerase chain reaction (PCR)

Amplifies low levels of specific DNA sequences; each cycle doubles the amount of specific DNA sequence.

Exponential accumulation of DNA fragment being amplified; defects in DNA appear as mutations.

Slightest trace of infection can be detected; more accurate than traditional tests for chlamydia; genetic disorders

Rate nephelometry

Measures either antigen or antibody in solution through the scattering of a light beam; antibody reagent used to detect antigen IgA, IgG, IgM; concurrent controls are run to establish amount of background scatter in reagents and test samples.

Light scatter proportionately increases as numbered size of immune complexes increases.

Quantitative immunoglobulins IgA, IgM, C-reactive protein, antistreptolysin O recorded in mg/dL or IU/mL

Flow cytometry

Blood cell types are identified with monoclonal antibodies (mABs) specific for cell markers by means of a flow cytometer with an argon laser beam; as the cells pass the beam, they scatter the light; light energy is converted into electrical energy cells and stained with green (fluorescence) or orange (phytoerythrin).

Light scatter identifies cell size and granularity of lymphocytes, monocytes, and granulocytes; color fluorochromes tagged to monoclonal antibodies bind to specific surface antigens for simultaneous detection of lymphocyte subsets.

Lymphocyte immunophenocytology differentiates B cells from T cells and T-helper cells from T-suppressor cells.

Restriction fragment length polymorphism (RFLP)

DNA-based typing technique

Epidemiology of nosocomial and communityacquired infections

cDNA probes

Uses cDNA probes directed against ribosomal RNA

Amplifies nucleic acid to identify presence of bacterial or viral load

Infectious disease such as tuberculosis, hepatitis C virus, and HIV

Serologic Versus Microbiologic Methods

Serologic testing for microbial immunology evaluates the presence of antibodies produced by antigens of bacteria, viruses, fungi, and parasites. The best means of establishing infectious disease etiology is by isolation and confirmation of the involved pathogen. Serologic methods can assist or confirm microbiologic analysis when the patient is tested late in the disease course, antimicrobial therapy has suppressed organism growth, or culture methods cannot verify a causative agent.


Syphilis Detection Tests

Syphilis is a venereal disease caused by Treponema pallidum, a spirochete with closely wound coils approximately 8 to 15 µm long. Untreated, the disease progresses through three stages that can extend over many years.

Antibodies to syphilis begin to appear in the blood 4 to 6 weeks after infection (Table 8.2). Nontreponemal tests determine the presence of reagin, which is a nontreponemal autoantibody directed against cardiolipin antigens. These tests include the rapid plasma reagin (RPR) and Venereal Disease Research Laboratory (VDRL) tests. The U.S. Centers for Disease Control and Prevention (CDC) recommend these tests for syphilis screening; however, they may show negative results in some cases of late syphilis. Biologic false-positive results can also occur (Table 8.3).

Conversely, treponemal (i.e., specific) tests detect antibodies to T. pallidum. These tests include the passive particle agglutination T. pallidum test (TP-PA) and the fluorescent treponemal antibody absorption test (FTA-ABS). These tests confirm syphilis when a positive nontreponemal test result is obtained. Because these tests are more complex, they are not used for screening. Certain states require automatic confirmation for all reactive screening tests by using a treponemal test such as the TP-PA or FTA-ABS.

Reference Values


Nonreactive, negative for syphilis

TABLE 8.2 Sensitivity of Commonly Used Serologic Tests for Syphilis



Primary (%)

Secondary (%)

Late (%)

Nontreponemal (Reagin) Tests

Venereal Disease Research Laboratory test (VDRL)




Rapid plasma reagin card test (RPR); automated reagin test (ART)




Specific Treponemal Tests

Fluorescent treponemal antibody absorption test (FTA-ABS)




Treponema pallidum particle agglutination (TP-PA)




(This new procedure has sensitivity similar to MHA-TP.)

* *Treated late syphilis.

Modified from Tramont EC: Treponema pallidum. In Mandell GI, Douglas RE, Bennett JE (eds): Principles and Practice of Infectious Diseases.

New York, John Wiley & Sons, 1985, p. 1329. Also product insert Serodia TP-PA, Fujirebio, Inc., Tokyo, Japan, 2000.

TABLE 8.3 Nonsyphilitic Conditions Giving Biologic False-Positive Results (BFPs) Using VDRL and RPR Tests


Approximate Percentage BFPs





Relapsing fever


Active immunization in children


Infectious mononucleosis


Lupus erythematosus


Lymphogranuloma venereum


Pneumonia, atypical


Rat-bite fever


Typhus fever




Infectious hepatitis


Leptospirosis (Weil’s disease)


Periarteritis nodosa










Rheumatoid arthritis


Rheumatic fever


Scarlet fever


Subacute bacterial endocarditis


Pneumonia, pneumococcal


Tuberculosis, advanced pulmonary


Blood loss, repeated

? (low)

Common cold

? (low)


? (low)

Sensitivity of FTA-ABS

Primary syphilis: 84% Secondary syphilis: 100% Latent syphilis: 100% Late syphilis: 96%

Sensitivity of TP-PA

Primary syphilis: 86% Secondary syphilis: 100% Latent syphilis: 100%

False-Positive and False-Negative Reactions

A positive reaction is not conclusive for syphilis. Several conditions produce biologic false-positive results for syphilis. Biologic false-positive reactions are by no means “false.” They may reveal the presence of other serious diseases. It is theorized that reagin (reaction) is an antibody against tissue lipids. Lipids are presumed to be liberated from body tissue in the normal course of activity. These liberated lipids may then induce antibody formation. Nontreponemal biologic false-positive reactions can occur in the presence of drug abuse, lupus erythematosus, mononucleosis, malaria, leprosy, viral pneumonia, recent immunization, or, on rare occasions, pregnancy. False-negative reactions may occur early in the disease course or during inactive or later stages of disease.

Interfering Factors

  • Hemolysis can cause false-positive results.

  • Hepatitis can result in a false-positive test.

  • Testing too soon after exposure can result in a false-negative test.

Lyme Disease Tests

Lyme disease is a multisystem disorder caused by the spirochete Borrelia burgdorferi. It is transmitted by the bite of tiny deer ticks, which reside on deer and other wild animals. Lyme disease is present worldwide, but certain geographic areas show higher incidences. Transmission to humans is highest during the spring, summer, and early fall months. The tick bite usually produces a characteristic rash, termed erythema chronicum migrans. If untreated, sequelae lead to serious joint, cardiac, and central nervous system (CNS) symptoms.

Serologic testing for antibodies to Lyme disease includes enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Antibody formation takes place in the following manner: Immunoglobulin M (IgM) is detected 3 to 4 weeks after Lyme disease onset, peaks at 6 to 8 weeks after onset, and then gradually disappears. IgG is detected 2 to 3 months after infection and may remain elevated for years. Current CDC recommendations for the serologic diagnosis of Lyme disease are to screen with a polyvalent ELISA (IgG and IgM) and to perform supplemental testing (Western blot) on all equivocal and positive ELISA results.

Western blot assays for antibodies to B. burgdorferi are supplemental rather than confirmatory because their specificity is less than optimal, particularly for detecting IgM. Two-step positive results provide supportive evidence of exposure to B. burgdorferi, which could support a clinical diagnosis of Lyme disease but should not be used as a criterion for diagnosis.

Reference Values


Negative for both IgG and IgM Lyme antibodies by ELISA and Western blot

Interfering Factors

  • False-positive results may occur with high levels of rheumatoid factors or in the presence of other spirochete infections, such as syphilis (cross-reactivity).

  • Asymptomatic individuals who spend time in endemic areas may have already produced antibodies to B. burgdorferi.

Legionnaires’ Disease Antibody Test

Legionnaires’ disease is a respiratory condition caused by Legionella pneumophila. It is best diagnosed by organism culture; however, the organism is difficult to grow.

Detection of L. pneumophila in respiratory specimens by means of direct fluorescent antibody (DFA) technique is useful for rapid diagnosis but lacks sensitivity when only small numbers of organisms are available. Serologic tests should be used only if specimens for culture are not available or if culture and DFA produce negative results.

Reference Values


Negative for legionnaires’ disease by indirect fluorescent antibody (IFA) test or ELISA

Chlamydia Antibody IgG Test

Chlamydia is caused by a genus of bacteria (Chlamydia spp.) that require living cells for growth and are classified as obligate cell parasites. Recognized species include Chlamydia psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis. C. psittaci causes psittacosis in birds and humans. C. pneumoniae is responsible for approximately 10% of cases of community-acquired pneumonia. C. trachomatis is grouped into three serotypes. One group causes lymphogranuloma venereum (LGV), a venereal disease. Another group causes trachoma, an eye disease. The third group causes genital tract infections different from LGV. Culture of the organism is definitive for chlamydiae. C. trachomatis infection is the most common reportable sexually transmitted infection (STI) in the United States. The national infection rate for C. trachomatis is estimated to be 3 million cases annually.

Because Chlamydia organisms are difficult to culture and grow, antibody testing aids in diagnosis of chlamydial infection.

Reference Values


Negative for chlamydia antibody by complement fixation (CF), IFA, and polymerase chain reaction (PCR) tests

Interfering Factors

Depending on geographic location, nonspecific titers can be found in the general healthy population.

Streptococcal Antibody Tests: Antistreptolysin O Titer (ASO), Streptozyme, Antideoxyribonuclease-B Titer (Anti-DNase-B, ADNase-B) (ADB, Streptodornase)

Group A β-hemolytic streptococci are associated with streptococcal infections or illness.

These tests detect antibodies to enzymes produced by organisms. Group A β-hemolytic streptococci produce several enzymes, including streptolysin O, hyaluronidase, and DNase B. Serologic tests that detect these enzyme antibodies include antistreptolysin O titer (ASO), which detects streptolysin O; streptozyme, which detects antibodies to multiple enzymes; and anti-DNase B (ADB), which detects DNase B. Serologic detection of streptococcal antibodies helps to establish prior infection but is of no value for diagnosing acute streptococcal infections. Acute infections should be diagnosed by direct streptococcal cultures or the presence of streptococcal antigens.

The ASO test aids in the diagnosis of several conditions associated with streptococcal infections, such as rheumatic fever, glomerulonephritis, endocarditis, and scarlet fever. Serial rising titers over several weeks are more significant than a single result. ADB antibodies may appear earlier than ASO in streptococcal pharyngitis, and this test is more sensitive for streptococcal pyoderma.

Reference Values


ASO titer:

Adult: <160 Todd units/mL or <200 IU

Child (5 to 12 years of age): 170-330 Todd units/mL Anti-DNase B (ADB): A negative test is normal.

Preschool-aged children: <60 Todd units/mL

School-aged children: <170 Todd units/mL

Adults: <85 Todd units/mL

Streptozyme: negative for streptococcal antibodies

Interfering Factors

  • An increased titer can occur in healthy carriers.

  • Antibiotic therapy suppresses streptococcal antibody response.

  • Increased B-lipoprotein levels inhibit streptolysin O and produce falsely high ASO titers.

Helicobacter pylori (HPY) IgG Antibody Serum, Stool, and Breath (PY) Test

H. pylori (previously known as Campylobacter pylori) is a bacterium associated with gastritis, duodenal and gastric ulcers, and possibly gastric carcinoma. The clinician orders this test when screening a patient for possible H. pylori infection. The organism is present in 95% to 98% of patients with duodenal ulcers and 60% to 90% of patients with gastric ulcers. A person with gastrointestinal symptoms with evidence of H. pylori colonization (e.g., presence of specific antibodies, positive breath test, positive culture, positive biopsy) is considered to be infected with H. pylori. A person without gastrointestinal symptoms having evidence of the presence of H. pylori is said to be colonized rather than infected.

This test detects H. pylori infection of the stomach. Traditionally, the presence of H. pylori has been detected through biopsy specimens obtained by endoscopy. As with any invasive procedure, there is risk and discomfort to the patient. Noninvasive methods of detection include the following:

  • Breath: measures isotopically labeled CO2 in breath specimens

  • Stool: H. pylori stool antigen test (HpSa)

The presence of H. pylori-specific IgG antibodies has been shown to be an accurate indicator of H. pylori colonization. ELISA testing relies on the presence of H. pylori IgG-specific antibody to bind to antigen on the solid phase, forming an antigen-antibody complex that undergoes further reactions to produce a color indicative of the presence of antibody and is quantified using a spectrophotometer or ELISA microweld plate reader. The sensitivity is 94% and specificity 78%, compared with an invasive procedure, such as biopsy, for which the sensitivity is 93% and specificity 99%.

Reference Values


Negative for H. pylori by ELISA indicates no detectable IgG antibody in serum or stool. A positive result indicates the presence of detectable IgG antibody in serum or stool.


Negative: <50 disintegrations per minute (DPM) for H. pylori 50-199 DPM indeterminate for H. pylori >200 DPM positive for H. pylori


Epstein-Barr Virus (EBV) Antibody Tests: Infectious Mononucleosis (IM) Slide (Screening) Test, Heterophile Antibody Titer, Epstein-Barr Antibodies to Viral Capsid Antigen and Nuclear Antigen

Epstein-Barr virus (EBV) is a herpesvirus found throughout the world. The most common symptomatic manifestation of EBV infection is a disease known as infectious mononucleosis (IM). This disease induces formation of increased numbers of abnormal lymphocytes in the lymph nodes and stimulates increased heterophile antibody formation. IM occurs most often in young adults who have not been previously infected through contact with infectious oropharyngeal secretions. Symptoms include fever, pharyngitis, and lymphadenopathy. EBV is also thought to play a role in the etiology of Burkitt’s lymphoma, nasopharyngeal carcinoma, and chronic fatigue syndrome.

The most common test for EBV is the rapid slide test (Monospot) for heterophile antibody agglutination. The heterophile antibody agglutination test is not specific for EBV and therefore is not useful for evaluating chronic disease. If the heterophile test is negative in the presence of acute IM symptoms, specific EBV antibodies should be determined. These include antibodies to viral capsid antigen (anti-VCA) and antibodies to EBV nuclear antigen (EBNA) using IFA and ELISA tests.

Diagnosis of IM is based on the following criteria: clinical features compatible with IM, hematologic picture of relative and absolute lymphocytosis, and presence of heterophile antibodies.

Reference Values


Negative for IM by latex agglutination

Negative for EBV antibodies by IFA or ELISA

Hepatitis Tests: Hepatitis A (HAV), Hepatitis B (HBV), Hepatitis C (HCV), Hepatitis D (HDV), Hepatitis E (HEV), Hepatitis G (HGV)

Hepatitis can be caused by viruses and several other agents, including drugs and toxins. Approximately 95% of hepatitis cases are due to five major virus types: hepatitis A, B, C, D, and E (Table 8.4). Diagnosing the specific virus is difficult because the symptoms (e.g., chills, weight loss, fever, distaste for cigarettes and food, darker urine and lighter stool) presented by each viral type are similar. Additionally, some individuals may be asymptomatic or have very mild symptoms that are ascribed to the “flu.” Serologic tests for hepatitis virus markers have made it easier to define the specific type.

Hepatitis A virus (HAV), which is acquired through enteric transmission, infects the gastrointestinal tract and is eliminated through the feces. Serologically, the presence of the IgM antibody to HAV (IgM anti-HAV) and the total antibody to HAV (total anti-HAV) identifies the disease and determines previous exposure to or recovery from HAV.

Hepatitis B virus (HBV) demonstrates a central core containing the core antigen and a surrounding envelope containing the surface antigen: less than 0.01 pg/mL for viral load. Detection of core antigen (HBcAg), envelope antigen (HBeAg), and surface antigen (HBsAg) or their corresponding antibodies constitutes hepatitis B serologic or plasma assessment. Viral transmission occurs through exposure to contaminated blood or blood products through an open wound (e.g., needle sticks, lacerations). Hepatitis monitoring panel for serial testing includes four B markers: HBsAg, HBeAg, anti-HBe, and anti-HBs. Interpretation depends on clinical setting. Hepatitis B DNA Ultra Sensitive Quantitative PCR is the most sensitive test available for hepatitis B viral load.

Hepatitis C virus (HCV), formerly known as non-A, non-B hepatitis, is also transmitted parenterally. HCV infection is characterized by presence of antibodies to hepatitis C (anti-HCV) and levels of
alanine aminotransferase (ALT) that fluctuate between normal and markedly elevated. Levels of anti-HCV remain positive for many years; therefore, a reactive test indicates infection with HCV or a carrier state but not infectivity or immunity. PCR or reverse transcriptase PCR (RT-PCR) (viral load), which detects HCV RNA, should be used to confirm infection when acute hepatitis C is suspected. A negative hepatitis C antibody (recombinant immunoblot assay [RIBA]) does not exclude the possibility of HCV infection because seroconversion may not occur for up to 6 months after exposure.

TABLE 8.4 Hepatitis Test Findings in Various Stages

Disease Stages

Viral Specific and Serologic Tests







IgM anti-HAV

IgM anti-HBc, HBsAg



IgM anti-HEV


Fecal HAV 1-2 wk before symptoms

2%-10% of all persons >5 yr will progress to chronic infection

85% Anti-HCV

6% total anti-HDV






Total anti-HDV




Anti-HBe, anti-HBs




Viral load (viral genome)



Carrier state




HBAg, anti-HDV



Total anti-HAV

Anti-HBs, total anti-HBc




Acute viral panel

IgM anti-HAV


Anti-HCV, HIV test also

Hepatitis D virus (HDV) is encapsulated by the HBsAg. Without the HBsAg coating, HDV cannot survive. Because HDV can cause infection only in the presence of active HBV infection, it is usually found where a high incidence of HBV occurs. Transmission is parenteral. Serologic HDV determination is made by detection of the hepatitis D antigen (HDAg) early in the course of the infection and by detection of anti-HDV antibody (anti-HDV) in the later stages of the disease.

Hepatitis E virus (HEV) is transmitted enterically and is associated with poor hygienic practices and unsafe water supplies, especially in developing countries. It is quite rare in the United States. Specific serologic tests include detection of IgM and IgG antibodies to hepatitis E (anti-HEV).

Hepatitis G virus (HGV) is transmitted by contaminated blood supply and is seen when HCV and HBV are detected together. See Table 8.5 for a summary of the features of the different hepatitis agents.

The following terms are used:

ALT (alanine aminotransferase): an enzyme normally produced by the liver; blood levels may

increase in cases of liver damage Anti-HBc: antibody to hepatitis B core antigen Anti-HBe: antibody to hepatitis B envelope antigen Anti-HBs: antibody to hepatitis B surface antigen

TABLE 8.5 Summary of Clinical and Epidemiologic Features of Viral Hepatitis Agents


Hepatitis A

Hepatitis B

Hepatitis C

Hepatitis D

Hepatitis E

Hepatitis G Scan With HBV + HCV

Incubation period

45-50 d

30-150 d

15-110 d

30-150 d

230-240 d










Children: 10%; adults: 70%-80%






Asymptomatic patients

Most children

Most children; adults: 50%

About 75%



Contaminated blood

Routes of transmission














Blood supply






















Chronic state


Adults: 6%-10%; children: 25%-50%; infants: 70%-90%





Case fatality rate


1.4% Liver cancer

1%-2% Liver cancer with HBV and HCV


1%-2% Pregnant women: 20%


Antibody: a Y-shaped protein molecule (immunoglobulin) in serum or body fluid that either neutralizes an antigen or tags it for attack by other cells or chemicals; acts by uniting with and firmly binding to an antigen. The prefix anti- followed by initials of a virus refers to specific antibody against the virus.

Chronic hepatitis: a condition in which symptoms or signs of hepatitis persist for >6 months

Cirrhosis: irreversible scarring of the liver that may occur after acute or chronic hepatitis

Delta agent: a unique RNA virus that causes acute or chronic hepatitis; requires HBV for replication and infects only patients who are HBsAg-positive; is composed of a delta antigen core and an

HBsAg coat; also known as HDV Endemic: present in a community at all times but occurring in a small number of cases

Enteric route: spread of organisms through the oral-intestinal-fecal cycle

Flavivirus: a family of small RNA viruses; HCV is similar to members of the Flavivirus family

Fulminant hepatitis: the most severe form of hepatitis; may lead to acute liver failure and death

HBcAg: hepatitis B core antigen

HBsAg: hepatitis B surface antigen

Hepatotropic: having an affinity for or exerting a specific effect on the liver

IgG: a form of immunoglobulin that occurs late in an infectious process

IgM: a form of immunoglobulin that occurs early in an infectious process

IgM anti-HAV: M-class immunoglobulin antibody to HAV

IgM anti-HBc: M-class immunoglobulin antibody to HBcAg

Immune globulin: a sterile solution of water-soluble proteins that contains those antibodies normally present in adult human blood; used as a passive immunizing agent against various viruses such as HAV

Negative-sense RNA virus: a virus in which the viral proteins are encoded by messenger RNA molecules that are complementary to the viral genome

New virusesGBV-A, GBV-B, and GBV-C: may be causative agents in non-A through E hepatitis

Non-A, non-B hepatitis: viral hepatitis caused by viruses other than A, B, or D (e.g., C, E)

Parenteral: entering the body subcutaneously, intramuscularly, or intravenously or other means whereby the organisms reach the bloodstream directly

Positive-sense RNA virus: a virus in which the parenteral (or genomic) RNA serves as the messenger RNA for protein synthesis

Recombinant antigen: an antigen that results from the recombination of genetic components, which then are artificially introduced into a cell, leading to synthesis of a new protein

Viral load: the amount or concentration of virus in the circulation

These measurements are used for differential diagnosis of viral hepatitis, viral load. Serodiagnosis of previous exposure and recovery of viral hepatitis is complex because of the number of serum or plasma markers necessary to determine the stage of illness. Testing methods include ELISA, microparticle enzyme immunoassay (MEIA), PCR, and RT-PCR and tests for viral genome (viral load).

Indications for Hepatitis A Vaccine

Pre-exposure Protection

  • Children should be vaccinated between 12 and 23 months of age.

  • Communities with existing vaccination programs for children 2 to 18 years of age should maintain their programs.

  • In areas without vaccination programs, catch-up vaccination of unvaccinated children 2 to 18 years of age can be considered.

Individuals at Increased Risk

  • Persons traveling to or working in countries that have high or intermediate endemicity

  • Users of injection and noninjection illicit drugs

  • Persons with clotting-factor disorders who have received solvent-detergent, treated high-purity factor VIII concentrates

  • Homosexual men

  • Individuals working with nonhuman HAV-infected primates

  • Food handlers

  • Persons employed in child care centers

  • Health care workers

  • Susceptible individuals with chronic liver disease

Indications for Hepatitis B Vaccine

  • Family members of adoptees from foreign countries who are HBsAg positive

  • Health care workers (dentist, DO, MD, RN, and trainees in health care fields)

  • Hemodialysis patients or patients with early renal failure

  • Household or sexual contacts of persons chronically infected with hepatitis B

  • Immigrants from Africa or Southeast Asia; recommended for children <11 years old and all susceptible household contacts of persons chronically infected with hepatitis B

  • Injection drug users

  • Inmates of long-term correctional facilities

  • Clients and staff of institutions for the developmentally disabled

  • International travelers to countries of high or intermediate HBV endemicity

  • Laboratory workers

  • Public safety workers (e.g., police, fire fighters)

  • Recipients of clotting factors. Use a fine needle (<23 gauge) and firm pressure at injection site for >2 minutes.

  • Persons with STIs or multiple sexual partners in previous 6 months, prostitutes, homosexual and bisexual men

  • Postvaccination blood testing is recommended for sexual contacts of HBsAg-positive persons; health care workers, recipients of clotting factors, those who are HBsAg-positive are at high risk.

  • Persons in nonresidential day care programs should be vaccinated if an HBsAg-positive classmate behaves aggressively or has special medical problems that increase the risk for exposure to blood. Staff in nonresidential day care programs should be vaccinated if a client is HBsAg-positive.

    • Observe enteric and standard precautions for 7 days after onset of symptoms or jaundice with hepatitis B. Hepatitis A is most contagious before symptoms or jaundice appears.

    • Use standard blood and body fluid precautions for type B hepatitis and B antigen carriers. Precautions apply until the patient is HBsAg negative and the anti-HBs appears. Avoid “sharps” (e.g., needles, scalpel blades) injuries. Should accidental injury occur, encourage some bleeding, and wash area well with a germicidal soap. Report injury to proper department, and follow up with necessary interventions. Put on gown when blood splattering is anticipated. A private hospital room and bathroom may be indicated.

  • Persons with a history of receiving blood transfusion should not donate blood for 6 months. Transfusion-acquired hepatitis may not show up for 6 months after transfusion. Persons who test positive for HBsAg should never donate blood or plasma.

  • Persons who have sexual contact with hepatitis B-infected individuals run a greater risk for acquiring that same infection. HBsAg appears in most body fluids, including saliva, semen, and cervical secretions.

  • Observe standard precautions in all cases of suspected hepatitis until the diagnosis and hepatitis type are confirmed.

Reference Values


  • Negative (nonreactive) for hepatitis A, B, C, D, or E by ELISA, MEIA, PCR, RIBA, or RT-PCR

  • Negative or undetected viral load (not used for primary infection, only to monitor). PCR requires a separate specimen collection.

  • Hepatitis B viral DNA (HBV-DNA) negative or nonreactive viral load (<0.01 pg/mL) in an infected individual before treatment

Human Immunodeficiency Virus (HIV-1/2) Antibody Tests, HIV Group O, Antibody to Human Immunodeficiency Virus (HIV-1/2); Acquired Immunodeficiency Syndrome (AIDS) Tests

These tests detect human immunodeficiency viruses types 1 and 2 (HIV-1/2), which cause AIDS. Infection with HIV-1 is most prevalent in the United States and Western Europe. Most cases associated with HIV-2 are reported in West Africa. Tests to detect the presence of HIV-1 antibody screen blood and blood products that will be used for transfusion and tissue and organs for transplantation. They are also used to test people at risk for developing AIDS, such as intravenous drug users, sexual partners of HIV-infected persons, and infants born to HIV-infected women (Table 8.9). The diagnosis of AIDS must be clinically established. Tests used to determine the presence of antibodies to HIV-1 include ELISA, Western blot, and PCR. PCR has been evaluated as a means to detect viral load by viral nucleic acid test (NAT) after infection but before seroconversion.

A single reactive ELISA test by itself cannot be used to diagnose AIDS. The test should always be repeated in duplicate using the same blood sample. If repeatedly reactive, follow-up tests using Western blot should be done. A positive Western blot is considered confirmatory for HIV. The combination HIV-1/2 test has replaced the HIV-1 test for screening blood and blood products for transfusion. It is also used for testing potential organ transplant donors.

TABLE 8.9 Diagnostic Testing for HIV

Whom to Test

How to Test

When to Test

Men who have sex with men, IV drug users, recreational drug users, those engaging in unprotected sex, those attending STD clinics, pregnant women, those with signs and symptoms of unusual pneumonia, skin lesions, mononucleosis-like syndrome; persons known to be infected with HIV

Screening EIA and confirmatory tests. Western blot confirmatory test detects antibodies to HIV-1 core antigens: gp41, gp120, gp160, p18, p24, p31, p40, p65, p55/51. IFA confirmatory test detects potent antibodies by fluorescein-tagged secondary antibodies. Viral RNA and p24 antigen are used along with CD4 count to monitor treatment. Nucleic acid amplification testing (NAT) to monitor “viral load.” Rapid testing: single-use diagnostic system (SUDS) results in 1 hour.

As early a detection as possible so that proper treatment, decrease in transmission, and modified behaviors can occur. Mother-to-child (vertical transmission) treated during pregnancy and delivery, and exposed infants within 48 hours of delivery; transmission of HIV can occur in utero, during birth, and by breast-feeding.

Reference Values


Negative for HIV antibodies against HIV antigens types 1 and 2 by ELISA, enzyme immunoassay (EIA), and Western blot HIV proviral RNA: not reactive or negative by PCR HIV proviral DNA: not reactive or negative HIV core P24 antigen: not reactive or negative NAT: Viral load is low.

Interfering Factors

  • Nonreactive HIV test results occur during the acute stage of disease when the virus is present but antibodies are not sufficiently developed to be detected. It may take up to 6 months for the test
    result to become positive. During this stage, the test for the HIV antigen may confirm an HIV infection (Fig. 8.1).

  • Test kits for HIV are extremely sensitive. As a result, nonspecific reactions may occur if the tested person has been previously exposed to HIV human cells or the growth media.

FIGURE 8.1. Time course for appearance of viral and serologic markers during primary HIV infection. (Modified from Busch MP, Satten GA. Time course of viremia and antibody seroconversion following human immunodeficiency virus exposure. Am J Med 1997;102(5B):117—124. Reproduced with permission of EXCERPTA MEDICA, INC. via Copyright Clearance Center.)


Rubella Antibody Tests

Rubella, a mild, contagious illness characterized by an erythematous maculopapular rash, is observed primarily in children 5 to 14 years of age and in young adults. The disease, commonly called German or 3-day measles, may be asymptomatic or may involve a 1- to 5-day prodromal period of malaise, headache, cold symptoms, low-grade fever, and suboccipital lymphadenopathy.

Although the illness is mild in children, it may cause the congenital rubella syndrome in the fetus of a mother infected early in pregnancy. As many as 85% of infants infected during the first 8 weeks of gestation have detectable defects by 4 years of age. The classic abnormalities associated with the rubella syndrome include congenital heart disease, cataracts, and neurosensory deafness. After 20 to 24 weeks of gestation, congenital abnormalities are rare.

The quantitative measurement of IgG antibodies to rubella virus aids in the determination of immune status. Assay results of 10 IU/mL of antibody are negative or not immune. Assay results >10 IU/mL are considered positive or immune. A positive result of IgM antibody indicates a congenital or recent infection. The measurement of IgM class antibodies for determination of acute-phase infection is recommended in all age groups. IgM rubella antibody determination is
usually not recommended when the patient is >6 months of age. Unlike IgG class antibodies, IgM antibodies are larger molecules and cannot cross the placenta, thus determining that the infant has an active form of the disease.

Reference Values


Negative for rubella IgG antibodies by ELISA or chemiluminescence (<7 IU/mL: no immunity)

Negative for IgM antibodies by ELISA or chemiluminescence (<0.9 IU/mL: no infection)

Positive for rubella IgG antibodies by ELISA or chemiluminescence (>10 IU/mL; immunity, indicates a current or previous exposure or immunization to rubella) Positive for rubella IgM antibodies (with or without positive IgG) by ELISA or chemiluminescence (>1.1 IU/mL; indicates a current or recent infection with rubella virus)

Measles (Rubeola) Antibody Tests

Classified as a paramyxovirus, measles produces a highly contagious respiratory infection. The disease is spread during the prodrome phase through direct contact with respiratory secretions in the form of droplets. Clinical infection with measles virus is characterized by high fever, cough, coryza, conjunctivitis, malaise, and Koplik’s spots on the buccal mucosa. An erythematous rash then develops behind the ears and over the forehead, spreading to the trunk.

Serology has become increasingly important as a tool for determining the immune status of the young adult population entering college or the military. In addition, the linkage between measles infection and premature delivery or spontaneous abortion supports screening pregnant mothers for susceptibility.

These tests determine susceptibility and immunity to measles virus. Since intensive immunization began in the United States in the 1970s, the incidence of measles infection has been reduced from approximately one half million cases annually (1960s) to fewer than 500 cases in recent years. Many individuals, however, may remain susceptible to measles virus because of vaccine failure or nonimmunization. A positive IgG coupled with a negative IgM result indicates previous exposure to measles virus and immunity to this viral infection. Positive IgM results, with or without positive IgG results, indicate a recent infection with measles virus.

Reference Values


Negative for measles IgG or IgM antibodies by ELISA: nonimmune

Positive for measles IgG antibody: immune; indicates a current or previous exposure or immunization to measles

Positive for measles IgM antibody (with or without positive IgG): indicates a recent infection with measles virus

Mumps Antibody Tests

The mumps virus (single-stranded RNA) is a member of the paramyxovirus group (genus Rubulavirus) and the etiologic agent of mumps in humans. Mumps is a generalized illness, usually accompanied by parotid (salivary gland) swelling and mild symptoms lasting 2 or more days. Parotitis as a presenting
symptom in mumps is usually sufficient to preclude confirmation by serology. However, one third of mumps infections are subclinical and may require viral isolation to confirm mumps infection. Infection with mumps virus, whether symptomatic or subclinical, is generally thought to offer lifelong immunity.

ELISA testing can be both specific and sensitive for the detection and measurement of serum proteins. Current methods for serodiagnosis of mumps include in vitro serum neutralization, hemagglutination inhibition (HAI), IFA, and CF. These test methods, however, lack specificity, which limits their usefulness in establishing immune status.

Reference Values


Negative for mumps IgG or IgM antibodies by ELISA: nonimmune Negative for viral antigen by RT-PCR

Positive for mumps IgG antibody: immune; indicates a current or previous exposure or immunization to mumps virus

Positive for mumps IgM antibody: indicates a current or recent infection

Varicella-Zoster (Chickenpox) Antibody Test

Varicella-zoster virus (VZV) is a herpesvirus and causes chickenpox with primary infection, a highly contagious disease characterized by widely spread vesicular eruptions and fever. The disease is endemic in the United States and most commonly affects children 5 to 8 years of age, although adults and younger children, including infants, may develop chickenpox. VZV infection in a pregnant woman may spread through the placenta to the fetus, causing congenital disease in the infant.

Although a primary infection results in immunity to subsequent chickenpox, the virus remains latent in the body. When it is reactivated, VZV causes shingles (herpes zoster). The incubation period is 10 to 24 days. Fever and painful localized vesicular eruptions of the skin along the distribution of the involved nerves are the most common clinical symptoms.

The sensitivity, specificity, and reproducibility of ELISA immunoassays are comparable to other serologic tests for antibody such as IFA, CF, and HAI. A positive IgG result, coupled with a positive IgM result, indicates a current infection with VZV.

Reference Values


Negative for varicella-zoster IgG or IgM antibodies by ELISA: nonimmune

Positive for varicella-zoster IgG antibody: indicates a current or previous infection; in the absence of current clinical symptoms, may indicate immunity Positive for varicella-zoster IgM antibody; indicates a current or recent infection

Cytomegalovirus (CMV) Antibody Test

Cytomegalovirus (CMV) is a ubiquitous human viral pathogen that belongs to the herpesvirus family. Infection with CMV is usually asymptomatic (up to 60 days) and can persist in the host as a chronic or latent infection. Cytomegalovirus has been linked with sexually transmitted infections. Blood banks routinely screen for CMV antibodies and report these as CMV-negative or CMV-positive.

This test determines the presence of CMV antibodies and is routinely done in congenitally infected newborns, immunocompromised patients, and sexually active persons who present with mononucleosis-like symptoms. Antibody results must be evaluated in the context of the patient’s current clinical symptoms and viral culture results. Tests to detect CMV antigen are available and aid in early detection. Viral culture confirms CMV infection.

Reference Values


Negative for CMV-specific IgG and IgM by ELISA

Herpes Simplex Virus (HSV) Antibodies (HSV-1 and HSV-2 Tests)

Two types of herpes simplex virus exist. Herpes simplex virus type 1 (HSV-1) causes orofacial herpes; type 2 (HSV-2) causes genital and neonatal herpes. Serologic differentiation is difficult; therefore, type-specific antibody tests are required.

These tests identify the herpes simplex infections. Human herpes simplex virus (HSV) is found worldwide and is transmitted by close personal contact. The clinical course is variable, and symptoms may be mild enough to go unrecognized. Major signs and symptoms include oral and skin eruptions, genital tract infections and lesions, and neonatal herpes. Herpes simplex is also common in individuals with immune system deficiencies (e.g., cancers, HIV/AIDS, chemotherapy treatment). HSV antibody testing is also widely used for bone marrow recipients and donors.

Reference Values


Negative for HSV-1 and HSV-2 by ELISA and IFA

Human T-Cell Lymphotropic Virus (HTLV-I/II) Antibody Test

This test detects antibodies to HTLV-I, a retrovirus associated with adult T-cell leukemia (ATL) and demyelinating neurologic disorders. The presence of HTLV-I antibodies in an asymptomatic person excludes that person from donating blood; however, this finding does not mean that leukemia or a neurologic disorder exists or will develop. Specimens with a positive test result by EIA are referred for Western blot. The results of Western blot are for investigational use only at the time of this printing.

Reference Values


Negative for HTLV-I/II antibodies by EIA and Western blot

Parvovirus B-19 Antibody Test

These antibody tests detect parvovirus B-19, the only parvovirus known to cause human disease. The B-19 virus destroys red blood cell precursor cells and interferes with normal red blood cell production. In young children, it is associated with erythema infectiosum, a mild, self-limited disease characterized by a low-grade fever and rash. Recently, it has been associated with aplastic crisis in patients with chronic hemolytic anemia and in immunodeficient patients who have bone marrow failure.

Reference Values


Negative for parvovirus B-19-specific IgG and IgM antibodies by ELISA and IFA

Rabies Antibody Tests

Rabies, a viral infection, is transmitted in the saliva of infected animals, such as bats, raccoons, skunks, cats, and dogs. Serologic testing is diagnostic for the presence of rabies in animals. It also indicates the degree of antibody responses to rabies immunization (e.g., for people who routinely work with animals).

Reference Values


IFA <1:16 or direct fluorescent antibody (DFA) examination of the animal brain for presence of the virus


Fungal Antibody Tests: Histoplasmosis, Blastomycosis, Coccidioidomycosis

Certain fungal species are associated with human respiratory diseases acquired by inhaling spores from sources such as dust, soil, and bird droppings. Serologic tests may be used for diagnosis. Fungal diseases are categorized as either superficial or deep. For the most part, superficial mycoses are limited to the skin, mucous membranes, nails, and hair. Deep mycoses involve the deeper tissues and internal organs. Histoplasmosis, coccidioidomycosis, and blastomycosis are caused by deep mycoses.

These tests detect serum precipitin antibodies and CF antibodies present in the fungal diseases of coccidioidomycosis, blastomycosis, and histoplasmosis. Coccidioidomycosis, also known as desert fever, San Joaquin fever, and valley fever, is contracted through inhalation of Coccidioides immitis spores found in dust or soil. Blastomycosis is caused by infection with organisms of the genus Blastomyces. Histoplasmosis is a granulomatous infection caused by Histoplasma capsulatum.

Reference Values


Negative for fungal antibodies CF titer: <1:8 Immunodiffusion: negative

Interfering Factors

  • Antibodies to fungi may be found in blood samples from apparently healthy people.

  • When testing for blastomycosis, cross-reactions with histoplasmosis may occur.

  • More than 50% of patients having active blastomycosis yield a negative result by CF.

  • Recent histoplasmosis skin tests must be avoided because they cause elevated CF test results, which may be due to the stimulation from the skin test and not the systemic infection.

Candida Antibody Test

Candidiasis is usually caused by Candida albicans and affects the mucous membranes, skin, and nails (see Candida Skin Test). Compromised individuals with depressed T-cell function are most likely to have invasive disease.

Identifying the Candida antibody can be helpful when the diagnosis of systemic candidiasis cannot be shown by culture or tissue sample. Clinical symptomatology must be present for the test to be meaningful. Tests used include immunodiffusion; counterimmunoelectrophoresis (CIE), which is particularly valuable on CSF and urine specimens; and latex agglutination for Candida antigen.

Reference Values


Negative for Candida antibodies by immunodiffusion (ID)

Interfering Factors

  • Approximately 25% of the normal population tests positive for the presence of Candida.

  • Cross-reaction can occur with latex agglutination testing in persons who have cryptococcosis or tuberculosis.

  • Positive results can occur in the presence of mucocutaneous candidiasis or severe vaginitis.

Aspergillus Antibody Test

The aspergilli, especially Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger, are associated with pulmonary infections and invasive fatal disease sequelae in immunosuppressed patients. Manifestations of Aspergillus infections include allergic bronchopulmonary disease, lung mycetoma, endophthalmitis, and disseminated brain, kidney, heart, and bone disease.

This test detects antibodies present in aspergillosis, primarily allergic bronchopulmonary disease, or fungus ball.

Reference Values


Negative for Aspergillus antibody by immunodiffusion <1:8 by CF

Cryptococcus Antibody Test

Cryptococcus neoformans, a yeast-like fungus, causes a lung infection thought to be acquired by inhalation. The organism has been isolated from several natural environments, especially where weathered pigeon droppings accumulate.

This test detects antibodies present in Cryptococcus infections. It appears that about 50% of patients who present with antibodies have a predisposing condition such as lymphoma or sarcoidosis
or are being treated with steroid therapy. Infection with C. neoformans has long been associated with Hodgkin’s disease and other malignant lymphomas. In fact, C. neoformans, in conjunction with malignancy, occurs to such a degree that some researchers have raised the question regarding the possible etiologic relation between the two diseases. Tests ordered for this disease include latex agglutination testing for antigens or antibodies.

Reference Values


Negative for Cryptococcus antibody by IFA or tube agglutination (TA) IFA test has a specificity of 77%. TA test has a specificity of 89%.

Jun 11, 2016 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on Immunodiagnostic Studies

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