Focus
How to increase precision in HPLC sample injection. Rheodyne's Tech Note 5 shows how you can maximize HPLC precision by using skillful sample injection techniques. The information is drawn from hundreds of closely controlled experiments using different types of sample injectors and different load ing methods. The 6-page summary of results is written to help both the beginner and the experienced chromatographer The experiments yielded some surprising answers to highly practical questions. One question: How much sample must you waste to fill a sample loop "completely"? (It's more than you are likely to think.) Another question: When you are reading a syringe to judge the amount of sample being loaded, how much sample can you inject without impairing accuracy? (It's less than you may think.) Get the answers to these and other salient questions now.
Send for Tech Note #5. Contact Rheodyne, Inc., PO. Box 996, Cotati, California 94928, U.S.A. Phone (707) 664-9050.
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tion with Biotech Research Laborato ries, Rockville, Md.; Litton Bionetics, Kensington, Md.; and Travenol/Genentech Diagnostics, Cambridge, Mass.) were awarded licenses by the govern ment allowing them access to HTLVIII for commercial development. Ac cording to John Petricianni, director of the division of blood and blood products at the FDA, "These com panies were selected not only for their potential in mass producing and dis tributing the test, but also for their potential in product improvement and refinement." Each of the five licensed manufac turers developed test kit configura tions, and all were approved for clini cal trials by the FDA last fall. As in formation about the tests became
Most of the current concern is over the specificity of the test—the ELISA's ability to identify healthy blood donors as negative for HTLV-ΓΠ antibody available, controversy over their accu racy and the social impact of HTLVIII screening began to mount. Accord ing to Petricianni, much of this con cern arose because the clinical data had not been presented by the manu facturers to anyone other than the FDA, selected Public Health Service personnel, and the FDA Advisory Committee. As a result, a public work shop was held at the National Insti tutes of Health in February, which Petricianni described as "an effort on our part to be as responsive as we can to legitimate concerns about the test in advance of licensing." At this meet ing, all five manufacturers presented details about their test configurations, along with the results of clinical trials. Although the ELISA configurations of the five manufacturers differ slight ly, all are capable of detecting HTLV-III antibody in blood. The per centage of confirmed AIDS patients with positive test results, a measure of the sensitivity of the tests, ranges from 99.6% for the FDA-licensed Electronucleonics assay to 82.0% for the Travenol/Genentech test (which is still in the developmental stage). The "false negatives," those samples that don't indicate the presence of HTLV-
THE LC CONNECTION COMPANY CIRCLE 183 ON READER SERVICE CARD 774 A ·
ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985
III antibody when it's expected to be there, have several suspected causes. Operator error, an antibody level below the detection limit of the assay (patients with advanced cases of AIDS sometimes have negligible amounts of antibody in their blood), recent infec tion (it is apparently possible for an individual to carry the virus for six months without developing detectable antibodies), and inadequate antibody response due to some other medical condition can all cause false negative results. The percentage of these false negatives is very low; however, consid ering that 12 million units of blood are donated each year, a significant amount of AIDS-contaminated blood could be missed by the screening pro cess. Individuals with a high risk of exposure to AIDS are being asked not to donate in an effort to minimize the possibility of such blood entering the blood supply. Most of the current concern, how ever, is over the specificity of the test—the ELISA's ability to identify healthy blood donors as negative for HTLV-III antibody. The percentage of ELISA positive results during clini cal trials, as reported by the manufac turers, ranges from 0.2% (Abbott) to 1.5% (Travenol/Genentech). These values do not include the nonrepeatable positives (the false positives), which range from 0.3% for the Elec tronucleonics test to 1.6% for Tra venol/Genentech, and were considered negative when calculating specificity. For each of the tests, 50-60% of the initial positive results do not repeat positive by either ELISA or western blot, a complicated technique that al lows identification of antibodies to specific proteins associated with the HTLV-III virus. The proportion of false positives, which can occur because of operator error, extremely high test sensitivity, or cross-reactivity to antibodies of other viruses not related to AIDS, is expected to be high in populations with a low incidence of the disease. Because the western blot is potentially more specific than the ELISA, it can be used to help eliminate false posi tives, and the Public Health Service recommends that all donor samples that test antibody positive by ELISA be checked by western blot. But be cause this is a time-consuming and ex pensive technique, additional means of eliminating false positives, other than repeating the original ELISA, would be useful. Electronucleonics, which is a large-scale supplier of HTLV-III virus to Gallo's group at NCI, has developed a "control plate," a microtiter plate coated not with in-
