For the determination of the percentage Ki67+ among T cells, 500000 cells were required and this determination could be performed in 69 of 80 (86%) of the sessions. Indeed, this assay was given priority over the HIV RNA assay. This approach was instrumental in demonstrating changes in proliferating (Ki67+) T-cell populations over the year following HAART initiation, compared with base-line levels. Proliferating populations were calculated from the percentage of Ki67+ T cells in cell suspensions obtained by lymph node aspiration as recently described . As shown on Fig. 4b, the present approach allowed the percentage proliferation among T cells at various times after initiating HAART to be determined and comparison of these values with values obtained at base line by excisional biopsy. The total number of proliferating T cells (CD3+) in the lymphoid tissue remained stable over the first year of treatment (P=0.90, one-way ANOVA), but actually reflected opposite changes of CD4 and CD8 proliferating cells. Numbers of proliferating CD4 T cells increased over time, a difference that almost reached statistical significance (P=0.059, one-way ANOVA). This was compensated for by a significant decrease in the numbers of proliferating CD8 T cells (P=0.006, one-way ANOVA).
For the determination of the HIV RNA copy number in the cell suspension, 500000 cells were required, and enough cells were available for this assay in 39 of 80 (49%) sessions. Examples of changes of HIV-1 RNA load in lymph node cells of four patients are shown in Fig. 5: three out of four patients had undetectable HIV RNA (<3copies/106lymph node cells) from 24 to 48 weeks after treatment initiation.
Among the 87 aspiration sessions mentioned in this report, no adverse event (haemorrhage, infection) was noticed.
The lymphoid tissue is the central anatomic compartment for HIV replication and pathogenesis. Therefore, it is important to gain access to lymphoid tissue specimens in order to improve the understanding of HIV pathogenesis and the efficacy of antiretroviral therapy. Although surgical lymph node biopsy is the conventional approach to sample lymphoid tissue, providing the largest amount of biological material, its invasive nature and cost limits its wide use. Thus, the development of alternative, less invasive, sampling procedures is very important to allow repeated sampling of lymphoid tissue for the longitudinal assessment of virus load and of the cell populations changes in response to therapy.
On the basis of the present results, ultrasound-guided aspiration appears to be a non-invasive, safe, and ad libitum repeatable procedure. We show that this technology provides lymphoid tissue cell suspensions in a vast majority of non-selected patients, and thus represents a striking improvement over finger-guided aspiration [8,11]. In these respects, it compares favourably with surgical biopsy, core needle biopsy which provided samples in about 50% of the patients in a recent study , or tonsil biopsy [4,5].
The lymph node sampling procedure we describe here was instrumental for the immunophenotype analysis of the mononuclear cells and allowed the quantification of cell-associated HIV RNA. Of note, experimental evidence was provided that both immunologic and virologic measures obtained in cell suspensions isolated by this technology were similar to those in cell suspensions obtained from excisional lymph node biopsies.
While CD4 T-cell percentages in aspiration and excision samples were very similar, CD8 T-cell percentages, although significantly correlated, were somewhat lower in aspiration samples compared with excision samples (see Fig. 2). The reason for this discrepancy is unclear. Of note however, the aspiration was performed taking care to avoid the lymph node medullary blood vessels from fear of blood contamination. The aspirated samples thus represent a preferential sampling of the lymph node cortical and paracortical area avoiding the medullary region. CD8 T cells are greatly increased in HIV-infected lymph nodes . It is conceivable that the proportion of CD8 T cells is not as increased at the site of the aspiration as compared with the whole lymph node when sampled by excisional biopsy. However, these slight differences do not seem to have a major impact on the analysis of the changes in CD4 T-cell percentage following HAART.
With regard to the determination of viral load, similar amounts of HIV RNA copies per 106 cells were measured in cell suspensions obtained by either approach. A two-fold difference was observed in patient 1003 with a higher viral load in the aspiration sample compared with the excisional sample. However, these differences are within the range of variability of the PCR assay. Compared with solid tissues biopsies, our procedure does not allow the extraction of RNA from whole tissue, but only from isolated cells. Lafeuillade et al.  have nicely demonstrated, extracting total tissue or cell suspensions obtained from excisional biopsies, that the pool of extracellular HIV-1 RNA is far greater (2-3 Logs) than that of lymph node mononuclear cells. Interestingly, the level of HIV-1 RNA found by these authors in lymph node mononuclear cells of drug-naive patients was similar (105-106copies/106 cells) to the level that we found in cell suspensions obtained by aspiration or excisional biopsies in the present study (see Table 1 and Fig. 5, time 0).
Limitations of ultrasound-guided lymph node aspiration include the technical skills required both for performing the aspiration and for the manipulation of samples containing small cell numbers, the lack of information on the lymph node architecture, and the variable, sometimes limited cell number yield. Indeed, the present approach generates a cell suspension, not a tissue fragment. Obviously, this precludes the assessment and evaluation of lymphoid tissue architecture that can be performed in lymph node, tonsil and rectal biopsies [2-5].
However, the analysis of the changes in viral load is certainly the most important information for evaluation of the effectiveness of antiretroviral therapy. The ultrasound-guided lymph node aspiration is a potentially very useful approach for these purposes. It is true that one or two aspirations may not provide the number of mononuclear cells necessary to perform both flow cytometry and determinations of viral load. It is important, however, to underscore that in the two clinical studies in which this procedure was applied  and , the priority was to assess the immunologic changes following HAART introduction. There are no technical or safety reasons to preclude the performance of multiple aspirations to obtain larger numbers of cells for both immunologic and virologic analyses.
In conclusion, evidence is provided that ultrasound-guided lymph node aspiration is an ad libitum repeatable procedure for the longitudinal monitoring of immunologic and virologic changes in lymph nodes following antiretroviral therapy.
1. Trepel F. Number and distribution of lymphocytes in man. A critical analysis. Klin Wschr
2. Pantaleo G, Graziosi C, Demarest JF, et al
. HIV infection is active and progressive in lymphoid tissue during the clinically latent stage of disease. Nature
3. Embretson J, Zupancic M, Ribas JL, et al
. Massive covert infection of helper T lymphocytes and macrophages by HIV during the incubation period of AIDS. Nature
4. Haase AT, Henry K, Zupancic M, et al
. Quantitative image analysis of HIV-1 infection in lymphoid tissue. Science
5. Cavert W, Notermans DW, Staskus K, et al
. Kinetics of response in lymphoid tissue to antiretroviral therapy in HIV-1 infection. Science
6. Cohen OJ, Pantaleo G, Holodniy M, et al
. Decreased human immunodeficiency virus type 1 plasma viremia during antiretroviral therapy reflects downregulation of viral replication in lymphoid tissue. Proc Natl Acad Sci USA
7. Tamalet C, Lafeuillade A, Fantini J, Poggi C, Yahi N. Quantification of HIV-1 viral load in lymphoid and blood cells: assessment during four-drug combination therapy. AIDS
8. Burgisser P, Spertini F, Suter C, Pagani JL, Meylan PRA. Monitoring the response to antiviral treatment in the lymph nodes of HIV-infected patients by serial fine needle aspiration. J Infect Dis
9. Wong JK, Günthard HF, Havlir DV, et al
. Reduction of HIV-1 in blood and lymph nodes following potent antiretroviral therapy and the virologic correlates of treatment failure. Proc Natl Acad Sci USA
10. Fleury S, De Boer RJ, Rizzardi GP, et al. Limited CD4+ T-cell renewal in early HIV-1 infection: effect of highly active antiretroviral therapy. Nature Med
11. Meylan PRA, Burgisser P, Weyrich-Suter C, Spertini F. Viral load and immunophenotype of cells obtained from lymph nodes by fine needle aspiration compared to peripheral blood cells in HIV-infected patients. J Acquir Immune Defic Syndr
12. Yerly S, Rutschmann OT, Opravil M, et al. Cell-associated HIV-1 RNA in blood as indicator of viral load in lymph nodes. J Infect Dis
1999, (in press).
13. Bart PA, Rizzardi GP, Tambussi G, et al. Quantitative normalization of CD4 cells in blood and lymph node of HIV-infected persons at early stage of chronic infection and no previous antiretroviral therapy treated with abacavir plus amprenavir. VI International Conference on Retroviruses and Opportunistic Infections.
Chicago, January-February 1999. [abstract 626].
14. Harris M, Patenaude P, Cooperberg P, et al
. Correlation of virus load in plasma and lymph node tissue in human immunodeficiency virus infection. J Infect Dis
15. Lafeuillade A, Poggi C, Tamalet C, Profizi N. Human immunodeficiency virus type 1 dynamics in different lymphoid tissue compartments. J Infect Dis
The members of the Swiss HIV Cohort Study are M.Battegay (Co-Chairman of the Scientific Board), E.Bernasconi, Ph.Bürgisser, M.Egger, P.Erb (Chairman of the Group ‚Laboratories‚), W.Fierz, M.Flepp (Chairman of the Group ‚Clinics‚), P.Francioli (President of the SHCS, Centre Hospitalier Universitaire Vaudois, CH-1011- Lausanne), H.J.Furrer, P.Grob, B.Hirschel (Chairman of the Scientific Board), L.Kaiser, B.Ledergerber, R.Malinverni, L.Matter, M.Opravil, F.Paccaud, G.Pantaleo, L.Perrin, W.Pichler, J.-C.Piffaretti, M.Rickenbach (Head of Data Center), P.Sudre, J.Schupbach, A.Telenti, P.Vernazza, R. Weber.
Keywords:© 1999 Lippincott Williams & Wilkins, Inc.
Ultrasound-guided; lymph node; needle aspiration; T-cell subsets; HAART; T-cell proliferation; non-invasive; lymph node; longitudinal study