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HIV-1 Western Blot

Test Code: 30817

Please contact Client Services to set up a Pre-transplant account to order this assay.

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Clinical Utility

The GS HIV-1 Western Blot Kit is an in vitro qualitative assay for the detection and identification of antibodies to Human Immunodeficiency Virus Type 1 (HIV-1) in human serum, plasma, or dried blood spots. It is intended for use with persons of unknown risk as an additional, more specific test on human serum, plasma, or dried blood spot specimens found to be repeatedly reactive using a screening procedure, such as Enzyme Linked Immunosorbent Assay (ELISA), and as an additional, more specific test for use with serum, plasma, or dried blood spot specimens obtained from subjects found to be reactive using rapid HIV-1 tests.

About HIV

The major etiologic agent of Acquired Immunodeficiency Syndrome (AIDS) is a retrovirus called Human Immunodeficiency Virus Type 1 (HIV-1).1-3 Enzyme immunoassays (EIA) to detect the presence of viralspecific antibodies to HIV-1 have been described by several investigators. Patients with AIDS and AIDS-related conditions exhibit a high prevalence of antibodies to HIV-1, and antibodies to HIV-1 have also been reported in virus positive, asymptomatic individuals.4,5

The original purpose of HIV-1 screening assays was to detect potentially infectious units of blood and to prevent these units from being used in transfusion or in the manufacture of blood products for transfusion. However, these highly sensitive tests have a relatively low positive predictive
value for populations with a low prevalence of HIV-1 infection.6 Some specimens may contain antibodies to HLA Class II histocompatibility antigens found on certain cell lines used to produce the virus for commercial applications, or specimens may react with bacterial contaminants associated with production of recombinant proteins.7,8 Other individuals who have had no known exposure to HIV-1 react with HIV-1 core proteins in the EIA for unknown reasons.9 Since the psychosocial and medical implications of a positive antibody test may be devastating, it is prudent to perform additional testing on such samples to further demonstrate the presence of antibodies specific to HIV-1.

The Western blot as described by Tsang, et. al.,10 is useful for elucidating the specificity of the antibody response to HIV-1.11-13 In the Western blot assay, disrupted proteins of HIV-1 are fractionated by electrophoresis according to molecular weight using a polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS). The resolved protein bands are transblotted to a nitrocellulose sheet. The nitrocellulose sheet is then cut into strips, which are reacted with serum, plasma, or dried blood spot specimens. If virus-specific antibodies are present, they bind to their corresponding viral protein bands. The bands are visualized by using a phosphataselabeled goat anti-human immunoglobulin conjugate, followed by a substrate for the enzyme. The presence of specific HIV-1 immunoglobulins in serum specimens is indicated by labeling of HIV-1-specific proteins on the strip. Recognized HIV-1 viral antigens produce bands at gp160, gp120, p65, p55, p51, gp41, p40, p31, p24, and p18.

A sample that is repeatedly reactive in the EIA and POSITIVE on the Western blot is presumed to be positive for antibodies to HIV-1.14 Individuals with positive tests should be referred for medical evaluation. A diagnosis of AIDS can be made only if an individual meets the case definition of AIDS established by the Centers for Disease Control.

Procedure

Purified, inactivated HIV-1 strain LAV grown in the CEM cell line is disrupted and electrophoretically resolved into bands. The proteins are transblotted onto nitrocellulose sheets, which are cut into strips. Samples are diluted in Specimen Diluent/Wash and applied to the nitrocellulose strip. If specific HIV-1 antibody is present, it binds to proteins resolved on the strip. Unbound sample is removed by washing. The phosphataselabeled conjugate is then added to the strip and allowed to incubate. The conjugate attaches to antibody already bound to viral proteins on the strip. Excess conjugate is removed by washing. Color Development Reagent is then added to the strip. Reaction sites, where enzyme-labeled antibody is bound, are identified by purple bands. The position and intensity of the bands are compared to reference strips developed using Positive Control sera.

Test performed by ARUP Laboratories 500 Chipeta Way, Salt Lake City, UT. This test has not been cleared or approved for diagnostic use by the U.S. Food and Drug Administration. See package insert for more information.

2-5 Business days from receipt of specimen.

Specimen Type Order Code CPT Code NY Approved Volume Assay Range Special Instructions
Plasma 30817 86689 Yes

50 µL (min 20 µL)

Qualitative

  • Collect whole blood in an EDTA, lavender top tube. Lithium Heparin, Sodium Citrate, or Sodium Heparin tubes are also accepted. Do not freeze whole blood.
  • Sample sent in original vacutainer tube can be shipped ambient or refrigerated, must be recieved within 7 days.
  • If not shipping in original container, centrifuge and transfer 50 µL (min 20 µL) plasma to screw top tube. Specimens shipped at ambient or refrigerated temperature must be received within 7 days of collection, or frozen.
Serum 30817 86689 Yes

50 µL (min 20 µL)

Qualitative

  • Collect whole blood in an gold, red, or red/gray tiger top tube. Do not freeze whole blood.
  • Sample sent in original vacutainer tube can be shipped ambient or refrigerated, must be recieved within 7 days.
  • If not shipping in original container, centrifuge and transfer 50 µL (min 20 µL) serum to screw top tube. Specimens shipped a ambient or refrigerated temperature must be received within 7 days of collection, or frozen.
Shipping

All specimens must be labeled with patient's name and collection date. A Viracor/VRL Eurofins Pre-Transplant test requisition form must accompany each specimen. Multiple tests can be run on one specimen. Ship specimens FedEx Priority Overnight® to: VRL Eurofins, 6933 S. Revere Parkway, Centennial, CO 80112.

Causes for Rejection

Whole blood frozen, specimens beyond their acceptable length of time from collection as listed in the specimen handling, or specimen types other than those listed.

Specimens are approved for testing in New York only when indicated in the Specimen Information field above. The CPT codes provided are based on VRL Eurofins' interpretation of the American Medical Association's Current Procedural Terminology (CPT) codes and are provided for informational purposes only. CPT coding is the sole responsibility of the billing party. Questions regarding coding should be addressed to your local Medicare carrier. VRL Eurofins assumes no responsibility for billing errors due to reliance on the CPT codes illustrated in this material.

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