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Interpretive Handbook

Test 36006 :
HIV-1 Antibody Confirmation by Western Blot, Serum

Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test

AIDS is caused by 2 known types of HIV. HIV type 1 (HIV-1) is found in patients with AIDS, AIDS-related complex, and asymptomatic infected individuals at high risk for AIDS. The virus is transmitted by sexual contact, exposure to infected blood or blood products, or from an infected mother to her fetus or infant. HIV type 2 (HIV-2) infection is endemic only in West Africa, and it has been identified in individuals who had sexual relations with individuals from that geographic region. HIV-2 is similar to HIV-1 in viral morphology, overall genomic structure, and its ability to cause AIDS.


Antibodies against HIV-1 and HIV-2 are usually not detectable until 6 to 12 weeks following exposure and are almost always detectable by 12 months. They may fall into undetectable levels (ie, seroreversion) in the terminal stage of AIDS when the patient’s immune system is severely depressed


Routine serologic screening of patients at risk for HIV-1 or HIV-2 infection usually begins with a HIV-1/-2 antibody screening test, which may be performed by various FDA-approved assay methods, including rapid HIV antibody tests, enzyme immunoassays, and chemiluminescent immunoassay methods. In testing algorithms that begin with these methods, supplemental or confirmatory testing should only be ordered for patients who are found to be reactive by routine HIV-1/-2 antibody screening tests or by rapid HIV antibody tests. However, for testing algorithms that begin with rapid HIV antibody tests, confirmatory antibody testing should be requested only for specimens that are repeatedly reactive by these methods according to assay manufacturers' instructions for use.

Useful For Suggests clinical disorders or settings where the test may be helpful

Supplemental testing for HIV-1 infection in patients with indeterminate or reactive HIV-1 antibody results (regardless of the HIV-2 antibody result) by FDA-approved HIV-1/-2 antibody differentiation tests

Interpretation Provides information to assist in interpretation of the test results

Positive HIV-1 antibody by Western blot results would show at least 2 of the 3 following bands: p24, gp41, and gp120/160. All initially positive supplemental or confirmatory HIV test results should be verified by submitting a second serum specimen for repeat testing. Such positive HIV test results are required under laws in many states in the United States to be reported to the departments of health of the respective states where the patients reside.


Negative HIV-1 antibody by Western blot results with reactive HIV-1/-2 antigen and antibody screening test and reactive HIV-1 antibody results from an HIV-1/-2 antibody differentiation test may be due to a) false-reactive screening test and false-reactive HIV-1 antibody differentiation test results (especially in the absence of signs, symptoms, or risk factors for HIV infection), or b) HIV-2 infection. Negative test results do not exclude the possibility of acute or early HIV-1 infection, when the humoral response of the infected individual has not yet generated the full complement of HIV-1-specific antibodies.


Indeterminate HIV-1 antibody by Western blot patterns can be due to many causes, including early HIV-1 infection, cross-reactive antibodies produced from recent non-HIV infections, hypergammaglobulinemic states, connective tissue disorders, and pregnancy (alloantibodies). Individuals suspected of having acute or early HIV-1 infection should undergo detection of HIV-1 RNA (HIVQU / HIV-1 RNA Quantification, Plasma) or HIV proviral DNA (PHIV / HIV-1 Proviral DNA Qualitative Detection by PCR, Blood).


Unreadable HIV-1 antibody by Western blot results indicate the presence of smear or blotches obscuring proper reading of the blot strip. Such findings indicate binding of nonspecific antibodies in the patient's serum to the blot.

Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances

The United States Association of Public Health Laboratories recommends verification of all first-time positive confirmatory test results prior to the diagnosis of HIV infection. A second serum specimen should be obtained from the patient and submitted for repeat testing to verify all such positive results.


Positive HIV antibody by Western blot results in infants at <18 months of age and born to HIV-infected mothers may indicate passive transfer of maternal HIV antibodies. Screening, supplemental or confirmatory serologic tests for HIV-1 or HIV-2 antibodies cannot distinguish between active neonatal HIV infection and passive transfer of maternal HIV antibodies in infants during the postnatal period (up to 18 months). Diagnosis of HIV infection in newborns and infants up to 18 months should be made by virologic tests such as detection of HIV RNA (HIVQU / HIV-1 RNA Quantification, Plasma) or HIV proviral DNA (PHIV / HIV-1 Proviral DNA Qualitative Detection by PCR, Blood).


This test should be ordered only on sera that are either indeterminate or reactive for HIV-1 antibodies (regardless of the HIV-2 antibody result) by FDA-approved HIV-1/-2 antibody differentiation tests.


Although hemolyzed serum specimens are acceptable for testing, this assay is not FDA-approved for testing cadaveric serum specimens.


Assay performance characteristics have not been established for the following specimen characteristics:

-Cadaveric specimens

-Presence of particulate matter

Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.



See HIV Serologic Interpretive Guide in Special Instructions for further interpretive information.

Clinical References Provides recommendations for further in-depth reading of a clinical nature

1. Constantine N: HIV antibody assays May 2006. In HIV InSite Knowledge Base (online textbook) Available from URL: http://hivinsite.ucsf.edu/InSite?page=kb-00&doc=kb-02-02-01

2. Centers for Disease Control and Prevention: Revised recommendations for HIV testing of adults, adolescents, and pregnant women in health-care settings. MMWR 2006;55(RR-6):1-17

3. Bennett B, Branson B, Delaney K, et al: HIV testing algorithms: a status report. Association of Public Health Laboratories, April 2009. http://www.aphl.org/aphlprograms/infectious/hiv/Documents/ID_2009April_HIV-Testing-Algorithms-Status-Report.pdf