HIV-1 Western blot - serum or plasma specimen

Collect

PREFERRED: Serum is the preferred specimen. Collect blood in a 4 ml Gold Serum Separator Tube-Clot Activator & Gel.

REMARK: Plasma is an acceptable alternative specimen. Blood can be collected in plastic tubes containing dipotassium EDTA with or without gel separator, sodium citrate, sodium heparin, and acid-citrate dextrose.

Volume Required

4 ml of blood

Minimum Required

2 ml of blood

Transport

A total of 2-4 ml of blood (for every 2-3 HIV and Hepatitis tests ordered) should be collected.

For the CHOP enterprise, submit blood specimens as soon as possible at room temperature to Central Laboratory Services.

For outside clients, it is recommended that serum or plasma specimens be removed from the clot, red blood cells, or separator gel as soon as possible after collection and before shipping specimens. When shipping serum or plasma specimens, package and label specimens in compliance with applicable state, federal, and international regulations covering the transport of clinical specimens and infectious substances. Specimens may be shipped at 2-8C (wet ice) or frozen (dry ice). Do not exceed the storage time limitations listed under the heading of Stability.

Stability

Serum or plasma specimens should be stored for no longer than 3 days at room temperature or 7 days at 2-8C following specimen collection and separation from clot, red blood cells, or separator gel. If a storage period greater than 7 days is anticipated, serum or plasma should be stored frozen at -20C or colder. No more than 3 freeze-thaw cycles should be performed on any sample prior to testing, and specimens should not be stored in frost-free freezers.

Processing

An HIV-1 Western blot is automatically ordered on all specimens that test repeatedy reactive for HIV-1/HIV-2 by the 4th generation antigen/antibody screening immunoassay in order to confirm the results.

Unacceptable conditions

Do not use serum or plasma specimens that are heat-inactivated, pooled, grossly hemolyzed, or contain obvious microbial contamination. Performance has not been established for the use of boody fluids other than human serum or plasma.

Specimen Handling

Unprocessed blood specimens should routinely be refrigerated at 4C upon arrival in the laboratory. One should remove the serum or plasma from clotted blood, red blood cells, or separator gel as soon as possible to avoid hemolysis. The best results are generally observed for serum or plasma specimens that are clear and nonhemolyzed. Lipemic, icteric, or hemolyzed specimens should be avoided when possible, and specimens with obvious microbial contamination should not be used. Specimens containing unremoved clots, red blood cells, or particulate matter may give inconsistent results and should be clarified by centrifugation before testing.

Days Performed

Tuesday-Wednesday (Requires 2 days to complete)

Reported

1 week

Reflex Testing

N/A

CPT

86689

Methodology

Immunoblot Assay

Disease Information

Utility:

The Western blot procedure has been widely accepted as a confirmatory method for HIV antibody. It is intended for use as an additional, more specific test for HIV-1 antibodies in persons of unknown risk whose serum or plasma specimens have been found to be repeatedly reactive using screening procedures such as an enzyme immunoassay. In the U.S., Western blotting is usually performed for confirmation of antibodies specific to HIV-1 since the prevalence of HIV-2 infection is low. HIV-2 specific Western blots are not routine done, but are normally available from commercial reference laboratories.

Interpretation

POSITIVE: A specimen that is positive/reactive in both the antigen/antibody screening test and the Western blot is presumed to be positive for antibody to HIV-1, indicating infection with this virus. Notable exceptions include situations of passively-acquired (e.g., mother-to-fetus) antibody or experimental vaccination. Antibodies to HIV-2 may also cross-react with the proteins of HIV-1 used in the Western blot. Therefore, individuals infected with HIV-2 may have reactive tests in the HIV-1 Western blot. Usually, however, the cross-reactivity is incomplete and results in an indeterminate test. Individuals with positive test results should be referred for medical evaluation.

NEGATIVE: A negative Western blot result suggests, but does not completely prove, that the person has not been exposed to HIV-1. However, such findings may provide useful information in the context of medical evaluation for which clinical information is available. Additional testing is normally warranted.

INDETERMINATE: Indeterminate Western blot results mean that the detection of antibodies to HIV-1 can neither be confirmed or ruled out and such results should not be used as the basis for diagnosis of HIV-1 infections. Reasons for indeterminate Western blots include (1) early seroconversion, (2) loss of antibody responses in patients with late-stage AIDS, (3) seroreversion in uninfected infants with maternal antibody, (4) HIV-2 infection, and (5) failure to mount an adequate immune response. Due to the uncertainty associated with indeterminate Western blots, additional testing is recommended.

Reference Values

NONREACTIVE or negative by Western blot procedure for antibody to HIV-1

Remarks

As of 15 May 2012, written informed consent for HIV testing is no longer required; documentation of verbal consent is adequate. Our hospital policies have been modified to conform with the revised Pennsylvania Act 148 (state statute that governs HIV testing) and the emphasis of the Commonwealth of Pennsylvania and Centers for Diseases Control and Prevention to provide routine HIV testing of individuals 13-65 years of age. This policy change applies to CHOP affiliated inpatient and outpatient care sites in Pennsylvania, New Jersey and Delaware. The new HIV-related policies can be found in the online Patient Care Manual. The patient or person consenting to the test must still be notified of the intent to test and be provided with HIV-related information. Two handouts are available in the Patient-Family Education Manual to provide information about HIV and HIV testing: "Understanding HIV: Testing and Prevention" and "HIV Testing is for Everyone". A competent minor, regardless of age, is permitted under Pennsylvania state law to consent to HIV testing and/or treatment services. Parental consent is not required. Otherwise, verbal consent can be obtained only from a biologic parent, an adoptive parent, legal guardian, or by a court order. Documentation in the medical record must show that verbal consent was obtained. Documentation of verbal consent for an HIV-related test can be accomplished through the Epic hospital computer system by selecting one of two certifications; either "I certify that the patient and/or personal representative have been given information about HIV testing and have verbally consented to the test" OR "I certify that HIV testing is required because of a life threatening medical condition or occupational exposure, and obtaining consent is not possible at this time".