Confocal Microscopic Detection of Human Immunodeficiency Virus RNA-Producing Cells Dorothy E. Lewis, Michael Minshall, Nelda R Wray, Stephen W. Paddock, Louis C. Smith, and Martin M. Crane
From the Departments of Microbiology and Immunology and Medicine, Center for AIDS Research, Y.4 Medical Center, Baylor College of Medicine, and Department of Epidemiology, School of Public Health, University of Texas Science Center, Houston; and Integrated Microscopy Resource, University of Wisconsin, Madison
AIDS is characterized by a depletion of CD4+cells as the disease progresses . Because these cells are crucial in the generation of immune responses, at least some of the immunodeficiency that characterizes the syndrome is likely to result from the destruction of these cells. One of the major mysteries is the paradoxic findingthat the immunodeficiency and clinical manifestations of human immunodeficiency virus (HIV) infectionare so detrimental eventhough few HIVproducing cells have been detected in the peripheral blood (1 in 1()4-1OS) . Plausible arguments for the CD4+ cellular depletion include hypothesesthat noninfected CD4+ cells are depleted by syncytia formation, by cytotoxic T cells, or by antibody-dependent cellular cytotoxicity [3-5]. Although these mechanisms exist in vitro, there is no convincing evidence that any of them plays a significantfactor in vivo . It is also possiblethat the peripheral blooddoesnot accurately reflectthe HIV status of the CD4+cells in other areas of the body. Recent evidence suggests that HIV DNA, as detected by polymerase chain reaction amplification, exists in ""1 in
Received 1 June 1990. Financial support: National Institutes of Health (AI-22549, HL-15648 to D.E.L.; HL-27341, HL-33750 to L.C.S.), Center for AIDS Research, VA Medical Center, Houston (to D.E.L., N.P.W.), and the Robert A. Welch Foundation (to L.C.S.). The Integrated Microscopy Resource in Madison is an NIH Biomedical Technology Resource (RR-570). Reprints or correspondence: Dr. Dorothy E. Lewis, Department of Microbiology andImmunology, BaylorCollegeof Medicine, One BaylorPlaza, M929, Houston, TX 77030. The Journal of Infectious Diseases 1990;162:1373-1378 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6206-0024$01.00
100-1000 CD4+ cells in some AIDS patients . In addition, Ho et ale  showedthat infectiousHIV can be isolated from an average of 1 in 400 peripheral blood mononuclear cells (PBMC) in patients with AIDS. In another study, HIV RNA was detected using slot-blothybridization in 39 (80%) of 49 HIV-infected individuals but from only 106 cells. In some cases it could be further diluted, suggesting either that many more cells were transcriptionally active or that the few infectedcells produced large amountsof RNA . No correlation with clinical diagnosis was found. To determine the actual proportions of HIV RNA-producing cells, in situ hybridization is required. In the original in situ hybridization studies by Harper et ale  7 of 14 PBMC samples from AIDS related complex (ARC)or AIDS patients had detectable numbers of infected cells but none had >0.01 % HIV-transcribing cells and most had fewer infected cells. Toexaminethe hypothesisthat failure of previous attempts to detect high levels of HIV RNA transcribing cells might be due to the insensitivity of the in situ measurement, we coupled a sensitive, standardized 3sS-based HIV RNA in situ hybridization technique with a new detection method, confocal laser scanning microscopy. Materials and Methods Patients. Sixty-five HIV-infected homosexual men (26 AIDS, 9 ARC, and 30 asymptomatic) were selected from patients followed bythe GeneralMedicine Clinic,VA MedicalCenter, Houston. HIV diagnosis and Karnofskyperformance status [9-11] were assigned by the attending physician. The Kamofsky performance score is a relativemeasureof the abilityof the patientto function . A score of 100meansthe patientis normal, 90 indicates minordiseasesymp-
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A central anomaly in the pathogenesis of AIDS is that few actively infected CD4+ cells (1 in 1()4-1()S) have been detected in the peripheral blood, even though dramatic depletion (often >90%) of CD4+ cells is the hallmark of disease progression. A sensitive, 3sS-based human immunodeficiency virus (HIV) RNA in situ hybridization technique was coupled with a new detection method, confocal laser scanning microscopy, to examine transcriptionally active HJ¥.infected cells from individuals at different disease stages. In 35 symptomatic HJ¥.infected individuals (AIDS and AIDS related complex), an average of 1 in 350 mononuclear cells produced HIV RNA. In contrast, in an asymptomatic group of 30 individuals, an average of 1 in 2000 mononuclear cells produced HIV RNA. These data, obtained using this improved detection method, suggest there are more HIV RNA-producing cells in HJ¥.infected individuals than previously reported. In addition, increased numbers of HIV transcribing cells were found to correlate with declining clinical condition as assessed by Karnofsky performance score. These data suggest that viremia per se may account for the pathologic consequences in HIV infection.
creased the area of the specimen scanned by the laser beam and thus increased the magnification of that area of the slide. The confocal technique was found to be most important when the background intensity was high. Statisticalanalysis. Differences among groups were examined at the 5% significance level by Kruskal-Wallis one-way analysis of variance for continuous variables and x?- for categorical variables . The Armitage trend test and associated X2 was used to assess trend .
Results Confocal microscopy. The principal advantage of the confocal microscope for evaluation of in situ hybridization is its ability to examine a precise plane of focus within the specimen. This effectively eliminates most of the background . With such microscopic detection systems as clarkfield microscopy, all silver grains within the emulsion including those above and below the plane of focus are cumulatively part of the image. The confocal image captures reflected light only from a shallow (tVO.5 p.m) plane of focus. When the silver grains, which accumulate in the emulsion over HIV RNApositive cells, are visualized, most of the background silver grains are excluded from the confocal image because they are randomly distributed throughout the emulsion. Figure 1 compares brightfield, darkfield, and confocal reflectance images of a specimen of HIV-infected cells and demonstrates the enhanced sensitivity of the confocal technique. Most investigators use darkfield microscopy to examine in situ hybridization which, in our hands, is adequate when compared with the confocal technique when the background intensity is low. Figure 2 compares techniques with cells from an uninfected person. The absence of background makes it easy to classify cells in this sample, even in the presence of artifactual material. Figure 3 shows that the confocal reflectance image of a sample with high background intensity is superior to the clarkfield image, thus increasing the certainty that the cell is positive. HIVRNAinpatients. In 65 specimens from HIV-infected subjects, HIV RNA was detected in ~1 cell/10,000 mononuclear cells from 71% of the patients including 17 of 26 AIDS patients, 7 of 9 ARC patients, and 22 of 30 asymptomatic HIVinfected individuals (table 1). Because no significant differences were found between the AIDS and ARC groups, the data were pooled to form a symptomatic group. There was a statistically significant difference between the symptomatic group and the asymptomatic group in terms of mean numbers of HIV RNA-positive cells per 10,000 mononuclear cells (symptomatic = 28 ± 43 or 1 in 350 cells, asymptomatic = 5 ± 9 or 1 in 2000 cells, P < .02). It was of interest that in 15 (43%) of 35 symptomatic HIV-infected patients (10 of 26 AIDS patients and 5 of9 ARC patients), >1 of every 1000 mononuclear cells expressed HIV RNA. In contrast, only 4 (13%) of the 30 asymptomatic patients had similar numbers of HIV RNA-expressing cells (P< .05). There was a statisti-
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toms, 80 that the patient has more severe disease symptoms but is able to work, and 70 that patients can care for themselves but are unable to work. In situ hybridization. Peripheral blood specimens were transported to Baylor College of Medicine within 2 h of acquisition. All patient and control samples were analyzed blindly with no knowledge of clinical condition. Five uninfected individuals were selected as controls. A sample of the peripheral blood was taken for total white blood cell and differential counts. A panel of monoclonal antibodies from Becton Dickinson (Mountain View, CA) was used to determine cellular phenotype including numbers of CD4+ cells using a whole blood lysis technique. Nine samples were split into two parts and submitted with separate codes to assess the reliability of the in situ procedure. Blood cells were separated over ficoll gradients, washed, counted, and deposited onto coated slides using a cytocentrifuge. The cells were fixed in 4% paraformaldehyde and stored in 70% ethanol at - 20°C before in situ hybridization. Control samples included HIVpositive and -negative tissue culture cells treated identically. In situ hybridization was performed using the method of Harper et al.  with reagents from Oncor (Gaithersburg, MD). The 35S-labeled antisense riboprobe is 1-2 kb (kilobase) long from HIV clone B4-10 and is contained in an Sp6 plasmid. The assay kit contains positive, negative, and sensitivity control slides. To ensure that hybridization of the HIV-l probe was to RNA and not DNA, slides were not subjected to denaturation so that DNA strand separation was prevented. Cytocentrifuge preparations of cells were fixed in 4 % paraformaldehyde and stored in 70 % ethanol at -20°C for as long as 6 months. Before in situ hybridization, slides were rinsed in 2 x saline sodium citrate (SSC). The proteins were then acetylated to prevent nonspecific binding, incubated for 10 min, and treated to glycinate aldehyde groups for 30 min. The slides were washed in 2x SSC, dehydrated in increasing concentrations of ethanol, and air dried. The hybridization mixture containing the 35S-labeled probe was added to each cell area in a humidified 52°C incubator, coverslipped, and incubated for 3 h. The slides were rinsed in 2x SSC and incubated in prewarmed 50 % formamide/2 x SSC (vol/vol) for 20 min at 52°C with frequent agitation, rinsed in 2x SSC four times, and treated with RNase to digest any single-stranded RNA. The slides were then rinsed, dehydrated in increased ethanol concentrations, and air dried. For autoradiography, the slides were transferred to a darkroom, dipped in melted emulsion, dried, transferred to black slide boxes, and exposed for 2-3 days. For developing, slides were warmed to room temperature and then placed in chilled developer, water, and chilled fixative. After being stained with Giemsa and mounted, the slides were examined by confocal laser scanning microscopy to determine the proportion of positive cells. Confocal microscopy. In situ hybridization slides were examined at the Integrated Microscopy Resource, Madison, WI, using a BioRad MRC-500 confocallaser scanning microscope. A manual method for counting the slides and verifying positivity was developed. A top edge of the cellular pattern on the slide was located. A representative brightfield using xlO magnification was counted, and then an appropriate number of fields was counted to reach 10,000 cells. This procedure usually took rv5 min. Care was taken to exclude artifactual material not associated with the cells. Positive cells were identified using both the brightfield and confocal reflectance images. Questionable cells were verified using the zoom feature, which de-
JID 1990;162 (December)
JID 1990;162 (December)
with HIV. A, Brightfield image. B, Confocal image. Bright areas of reflection are associated with dark grains in A. C-E, In focus brightfield, confocal, and darkfield images of same field. In E, bright area at top right was associated with an artifact in the glass that was not observed in the confocal image because of the narrow depth of focus. All images taken at X 10.
cally significant (P < .001) difference in CD4+ cellular numbers in the clinical groups as reported previously . Finally, if only CD4+ lymphocytes are Hlv-infected in the peripheral blood as Schnittman et al. suggest , the possible percentage of CD4+ cells producing HIV RNA in our symptomatic groups (AIDS and ARC) would be 8 %; however,