title: Quality monitoring of HIV-1-infected and uninfected peripheral blood mononuclear cell samples in a resource-limited setting.
abstract: Human immunodeficiency virus type 1 (HIV-1) vaccine and natural history studies are critically dependent on the ability to isolate, cryopreserve, and thaw peripheral blood mononuclear cell (PBMC) samples with a high level of quality and reproducibility. Here we characterize the yield, viability, phenotype, and function of PBMC from HIV-1-infected and uninfected Ugandans and describe measures to ascertain reproducibility and sample quality at the sites that perform cryopreservation. We have developed a comprehensive internal quality control program to monitor processing, including components of method validation. Quality indicators for real-time performance assessment included the time from venipuncture to cryopreservation, time for PBMC processing, yield of PBMC from whole blood, and viability of the PBMC before cryopreservation. Immune phenotype analysis indicated lowered B-cell frequencies following processing and cryopreservation for both HIV-1-infected and uninfected subjects (P < 0.007), but all other major lymphocyte subsets were unchanged. Long-term cryopreservation did not impact function, as unstimulated specimens exhibited low background and all specimens responded to staphylococcal enterotoxin B (SEB) by gamma interferon and interleukin-2 production, as measured by intracellular cytokine staining. Samples stored for more than 3 years did not decay with regard to yield or viability, regardless of HIV-1 infection status. These results demonstrate that it is possible to achieve the high level of quality necessary for vaccine trials and natural history studies in a resource-limited setting and provide strategies for laboratories to monitor PBMC processing performance.
authors.affiliation: Makerere University Walter Reed Project, Kampala, Uganda.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Olemukan
authors.forename: Leigh Anne
authors.initials: LA
authors.lastname: Eller
authors.forename: Benson J
authors.initials: BJ
authors.lastname: Ouma
authors.forename: Ben
authors.initials: B
authors.lastname: Etonu
authors.forename: Simon
authors.initials: S
authors.lastname: Erima
authors.forename: Prossy
authors.initials: P
authors.lastname: Naluyima
authors.forename: Denis
authors.initials: D
authors.lastname: Kyabaggu
authors.forename: Josephine H
authors.initials: JH
authors.lastname: Cox
authors.forename: Johan K
authors.initials: JK
authors.lastname: Sandberg
authors.forename: Fred
authors.initials: F
authors.lastname: Wabwire-Mangen
authors.forename: Nelson L
authors.initials: NL
authors.lastname: Michael
authors.forename: Merlin L
authors.initials: ML
authors.lastname: Robb
authors.forename: Mark S
authors.initials: MS
authors.lastname: de Souza
authors.forename: Michael A
authors.initials: MA
authors.lastname: Eller
mesh_terms: D001793:Blood Preservation / Q000592:standards*; D001800:Blood Specimen Collection / Q000592:standards*; D002470:Cell Survival; D015925:Cryopreservation / Q000592:standards*; D016207:Cytokines / Q000378:metabolism; D003906:Developing Countries; D015658:HIV Infections / Q000276:immunology / Q000821:virology; D015497:HIV-1 / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D016130:Immunophenotyping; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000821:virology; D008213:Lymphocyte Activation; D011786:Quality Control; D013997:Time Factors; D014454:Uganda
chemical_list: D016207:Cytokines