A STUDY OF 2 PROCEDURES OF HIV-1 ISOLATION FROM WHOLE-BLOOD CULTURES

Culture techniques for isolation of HIV-1 from small amounts of whole blood (WE) treated with anticoagulant have been reported and gave resu lts identical to those of culture of separated peripheral blood mononu clear cells. Some authors obtained much higher isolation rates when ED TA was used instead of heparin. We compared two previously described t echniques for cultivation of HIV-1 from WB of adult HIV+ patients stag ed according to the CDC classification, In addition, we assessed the i nfluence of the type of anticoagulant used for the collection of blood in viral replication in cell cultures from whole blood, Small volumes of WE treated with either heparin or EDTA were cocultivated with phyt ohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PH A-PBMC) from healthy donors, We used two procedures for WE culture: pr ocedure I, based on the culture of 250 mu l of WB with 1 x 10(6) PHA-P BMC from donors; and procedure II based on the culture of 500 mu l of WB with 4x10(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 wi th fresh medium. In procedure II, one million fresh PHA-PBMC from dono rs was added on the 7th day of culture. The culture supernatant was as sayed 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 patien ts were similar using procedures I and II. In 8 HIV+ patients the isol ation rate was higher with heparin- than with EDTA-treated samples. Th e 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 is olate HIV-1 in 1 patient with procedure I, whereas we succeeded with p rocedure II. In stage II, HIV-I was isolated in 1 of 4 patients with b oth methods. HIV was isolated in cultures of WB from patients receivin g zidovudine or related nucleoside analogues and in cultures of WB fro m 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 CD 4+ T lymphocytes. Our results suggest that procedure II is more sensit ive than procedure I and that heparin is better than EDTA for collecti ng WB. We showed that the rate of HIV-1 isolation from WB increased in advanced-stage patients, Further studies are needed to define the cli nical applications of WB culture.