Shen L, Hober D, Benyoucef S, Ajana F, Gérard Y, Lion G, Vermersch A, Bocket-Mouton L, Mouton Y, Wattré P
Laboratoire de Virologie, Bât IRFPPS, CHU, Lille, France.
Microbiol Immunol. 1996;40(3):195-200. doi: 10.1111/j.1348-0421.1996.tb03334.x.
Culture techniques for isolation of HIV-1 from small amounts of whole blood (WB) treated with anticoagulant have been reported and gave results identical to those of culture of separated peripheral blood mononuclear cells. Some authors obtained much higher isolation rates when EDTA was used instead of heparin. We compared two previously described techniques for cultivation of HIV-1 from WB of adult HIV+ patients staged according to the CDC classification. In addition, we assessed the influence of the type of anticoagulant used for the collection of blood in viral replication in cell cultures from whole blood. Small volumes of WB treated with either heparin or EDTA were cocultivated with phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PHA-PBMC) from healthy donors. We used two procedures for WB culture: procedure I, based on the culture of 250 microliters of WB with 1 x 10(6) PHA-PBMC from donors; and procedure II based on the culture of 500 microliters of WB with 4 x 10(6) PHA-PBMC from donors. The cocultures were placed in 24-well plates and incubated for as long as 28 days in medium containing interleukin 2 (IL-2). Twice weekly half of the medium was replaced with fresh medium. In procedure II, one million fresh PHA-PBMC from donors was added on the 7th day of culture. The culture supernatant was assayed for the presence of HIV-1 p24 antigen in an enzyme immunoassay. The kinetics of HIV-1 replication in cultures of WB from 7 AIDS patients were similar using procedures I and II. In 8 HIV+ patients the isolation rate was higher with heparin- than with EDTA-treated samples. The isolation rate was higher in AIDS patients (n = 8) than in others with both methods. In stage IV patients without AIDS (n = 8) we failed to isolate HIV-1 in 1 patient with procedure I, whereas we succeeded with procedure II. In stage II, HIV-I was isolated in 1 of 4 patients with both methods. HIV was isolated in cultures of WB from patients receiving zidovudine or related nucleoside analogues and in cultures of WB from untreated patients. HIV-1 could not be isolated from WB of patients with more than 400 CD4+ T lymphocytes in their peripheral blood (n = 4); however, it was isolated from 14 of 16 patients with less than 400 CD4+ T lymphocytes. Our results suggest that procedure II is more sensitive than procedure I and that heparin is better than EDTA for collecting WB. We showed that the rate of HIV-1 isolation from WB increased in advanced-stage patients. Further studies are needed to define the clinical applications of WB culture.
已有报道称可从用抗凝剂处理过的少量全血（WB）中分离出HIV-1的培养技术，其结果与分离的外周血单个核细胞培养结果相同。一些作者使用乙二胺四乙酸（EDTA）而非肝素时获得了更高的分离率。我们比较了两种先前描述的从根据疾病控制与预防中心（CDC）分类分期的成年HIV阳性患者的全血中培养HIV-1的技术。此外，我们评估了用于采集血液的抗凝剂类型对全血细胞培养中病毒复制的影响。将用肝素或EDTA处理过的少量全血与来自健康供体的经植物血凝素（PHA）刺激的外周血单个核细胞（PHA-PBMC）共同培养。我们使用两种全血培养程序：程序I，基于将250微升全血与来自供体的1×10⁶个PHA-PBMC进行培养；程序II基于将500微升全血与来自供体的4×10⁶个PHA-PBMC进行培养。将共同培养物置于24孔板中，并在含有白细胞介素2（IL-2）的培养基中孵育长达28天。每周两次将一半培养基换成新鲜培养基。在程序II中，在培养的第7天添加来自供体的100万个新鲜PHA-PBMC。在酶免疫测定中检测培养上清液中HIV-1 p24抗原的存在。使用程序I和II时，来自7名艾滋病患者的全血培养物中HIV-1复制的动力学相似。在8名HIV阳性患者中，肝素处理的样本比EDTA处理的样本分离率更高。两种方法在艾滋病患者（n = 8）中的分离率均高于其他患者。在无艾滋病的IV期患者（n = 8）中，使用程序I时1名患者未分离出HIV-1，而使用程序II时成功分离出。在II期，两种方法在4名患者中的1名中分离出了HIV-1。在接受齐多夫定或相关核苷类似物治疗的患者的全血培养物以及未治疗患者的全血培养物中均分离出了HIV。无法从外周血中CD4⁺T淋巴细胞超过400个的患者的全血中分离出HIV-1（n = 4）；然而，从16名CD4⁺T淋巴细胞少于400个的患者中的14名中分离出了HIV。我们的结果表明程序II比程序I更敏感，并且肝素在采集全血方面优于EDTA。我们表明晚期患者中从全血分离HIV-1的比率增加。需要进一步研究来确定全血培养的临床应用。
