Nicholson JK, Bergeron M, Mandy F; International Conference on AIDS.

Int Conf AIDS. 1996 Jul 7-12; 11: 257 (abstract no. Th.A.4021).

Immunology Branch, Division of HIV/AIDS, NCID, CDC, Atlanta, GA, USA. Fax: (404) 639-2108.

Objective: Blood samples more than 24 hours old from HIV+ patients often pose a difficult challenge for immunophenotyping CD4+ cells. We evaluated 3 different gating strategies for immunophenotyping blood samples to determine which was the best for correctly identifying CD4+ cells in specimens of poor quality. Method: The 3 gating protocols evaluated were: (1) A homogeneous light scatter gate with corrections for lymphocyte purity (recommended by CDC and NIAID), and two heterogeneous protocols (2) T-cell gating (CD3+), and (3) Bright CD45 gating (recommended by NIAID). The latter 2 gates are based on fluorescence and side scatter characteristics. Fresh blood was obtained from at least 70 HIV+ and 29 HIV- donors. The 2-color samples were processed by the established laboratory protocol (QPrep or FACS Lysing system) for CD4 and CD8 determinations. 3-color tubes contained CD45/CD3/CD4, and CD45/CD3/CD8. Replicate specimens were spiked with unlysed whole blood to simulate poor specimen quality. Samples were analyzed on an EPIC-XL or FACScan instrument. Result: CD4 and CD8 T-cell results from spiked specimens that were analyzed through a light scatter gate were unacceptable (r2 less than 0.71) because of excess red cell contamination in the gate. Using T-cell or CD45 gate strategy gave results with a correlation of greater than 0.95. Conclusion: A T-cell or CD45 gate are preferred for specimens of poor quality (excess unlysed red cells). A light scatter gate is not acceptable for such specimens because the red cells cannot be resolved from the lymphocytes, and this variable contamination cannot be appropriately corrected. The comparison of two heterogeneous gating techniques to the conventional light scatter gate demonstrated the advantage of three-color immunophenotyping.

Publication Types:

• Meeting Abstracts

Keywords:

• AIDS Vaccines
• Acquired Immunodeficiency Syndrome
• Antigens, CD3
• Antigens, CD4
• Antigens, CD45
• Antigens, CD8
• CD4-Positive T-Lymphocytes
• CD8-Positive T-Lymphocytes
• Centers for Disease Control and Prevention (U.S.)
• Color
• Evaluation Studies
• Flow Cytometry
• HIV Infections
• HIV Seropositivity
• Humans
• Immunophenotyping
• Light
• Lymphocytes
• T-Lymphocytes
• immunology
• methods

Other ID:

• 96924434

UI: 102220333

From Meeting Abstracts