Janet K. A. Nicholson

Idiopathic CD4+ T-Lymphocytopenia -- An Analysis of Five Patients with Unexplained Opportunistic Infections

BACKGROUND Although patients with idiopathic CD4+ T-lymphocytopenia and serious opportunistic infections have been described previously, the clinical and immunologic features of this condition have not been well defined. METHODS We studied in detail five patients with idiopathic CD4+ T-lymphocytopenia. The studies included serologic testing, culture, and polymerase chain reaction for the human immunodeficiency virus (HIV) types 1 and 2, serologic testing for the human T-cell lymphotropic virus (HTLV) types I and II, lymphocyte phenotyping, immunoglobulin quantitation, and lymphocyte-transformation assays, as well as attempts to isolate a retroviral agent. The results were compared with those in HIV-infected persons matched for CD4+ T-cell counts and with those in normal controls. We also studied the spouses of patients and the blood donors for one patient. RESULTS In these five patients, there was no evidence of either HIV or HTLV infection. All the patients had both low percentages and low counts of CD4+ T cells, with relative increases in percentages, but not counts, of CD8+ cells. Numbers of B cells and natural killer cells were generally normal. As compared with HIV-infected persons, our patients had lower percentages and counts of CD8+ cells and more lymphopenia. CD4+ counts were relatively stable over time. Instead of the high immunoglobulin levels seen in HIV infection, these patients had normal or slightly low levels of immunoglobulins. The lymphocyte-transformation response to mitogens and antigens was depressed. Results in spouses and blood donors were normal. CONCLUSIONS Idiopathic CD4+ T-lymphocytopenia differs from HIV infection in its immunologic characteristics and in its apparent lack of progression over time. Nothing about the immunologic or viral-culture studies performed in these patients or about their family members or blood donors suggests that a transmissible agent causes this condition.

Evaluation of TruCount absolute-count tubes for determining CD4 and CD8 cell numbers in human immunodeficiency virus-positive adults.

ABSTRACT A single-platform technology that uses an internal bead standard and three-color flow cytometry to determine CD4 and CD8 absolute counts was evaluated for reproducibility and agreement. Values obtained using TruCount absolute-count tubes were compared to those obtained using a two-color predicate methodology. Sixty specimens from human immunodeficiency virus type 1-infected donors were shipped to five laboratories. Each site also analyzed replicates of 14 human immunodeficiency virus type 1-infected local specimens at 6 h and again at 24 h. The interlaboratory variability was significantly less with TruCount (median difference in percent coefficient of variation [%CV] between the two methods was −8% and −3% for CD4 and CD8, respectively) than with the predicate method. Intralaboratory variability was smaller, with a median difference in %CV of −1% for both CD4 and CD8 with 6-h samples and −2% and −3% for CD4 and CD8, respectively, with 24-h samples. Use of TruCount for shipped samples resulted in a median CD4 count change of 7 cells (50th estimated percentile) when all laboratories and CD4 strata were combined. For on-site samples, the median CD4 count change was 10 CD4 cells for 6-h samples and 2 CD4 cells for 24-h samples. Individual site biases occurred in both directions and cancelled each other when the data were combined for all laboratories. Thus, the combined data showed a smaller change in median CD4 count than what may have occurred at an individual site. In summary, the use of TruCount decreased both the inter- and intralaboratory variability in determining absolute CD4 and CD8 counts.

Exposure to Human T-Lymphotropic Virus Type III/Lymphadenopathy-Associated Virus and Immunologic Abnormalities in Asymptomatic Homosexual Men

Immunologic and serologic studies were done in 120 homosexual men who reportedly had no symptoms related to the acquired immunodeficiency syndrome. Forty-nine men (41%) had antibody to the retrovirus human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV), and 37 (31%) had an abnormal T-cell subset ratio and other immunologic abnormalities. These abnormalities were almost exclusively confined to men seropositive for the retrovirus. Multivariate statistical analysis showed that exposure to HTLV-III/LAV was the single best predictor of T-cell (and other) immunologic abnormalities. Sexual practice, particularly receptive anal intercourse, predicted exposure to HTLV-III/LAV but was not independently related to T-cell abnormalities. The association of other microbial serologic findings with HTLV-III/LAV seropositivity or T-cell changes was a coincidental function of sexual activity. Immunologic abnormalities in asymptomatic homosexual men are mostly related to exposure to HTLV-III/LAV.

Immune complexes in the acquired immunodeficiency syndrome (AIDS): Relationship to disease manifestation, risk group, and immunologic defect

Immune complex assays (and other immunologic tests) were performed on sera from 162 patients with the acquired immunodeficiency syndrome (AIDS) and 275 AIDS-related subjects. Immune complexes were detected in 89% of AIDS patients and 93% of homosexual men with lymphadenopathy. Immune complex levels in AIDS patients were not associated with a particular risk group or with types of opportunistic infection or malignancy; however, they correlated with other laboratory features of the immune defect (depression in T helper cells and T helper/suppressor-cell ratio, and IgG levels). Immune complexes were also detected in a lesser proportion of risk-group controls (homosexual men, hemophiliacs, Haitians). In risk-group controls, immune complex levels were associated with certain features reflecting sexual practice, blood product exposure, or infection, but these features did not account for the higher levels found in AIDS patients. In appropriate situations, immune complex assays may be of value as screening tests or, possibly, as prognostic indicators for AIDS or AIDS-related syndromes.

Three-color supplement to the NIAID DAIDS guideline for flow cytometric immunophenotyping.

Since the publication of the ‘‘NIAID DAIDS Guideline for Flow Cytometric Immunophenotyping’’ (1) in 1993, significant scientific and technological advances in the development and production of reagents, instrumentation, and software have permitted widespread access to multicolor flow cytometry in both research and clinical laboratories. As is often the case with complex new technologies, each advance, while opening up exciting new capabilities, can also bring new sources of variability and a commensurate increase in the requirements for quality assurance at all levels. The purpose of this document is to update the 1993 NIAID DAIDS Guideline with new recommendations designed to help standardize the methodology and minimize measurement variability in determining patients’ CD4 and CD8 T-cell counts using 3-color flow cytometry. Issues pertaining to specimen collection and handling, problematic specimens, hematology, and laboratory performance assessment were addressed in the previous guideline.1 In assembling this supplement, factors such as reduced technician time, decreased need for isotype controls, fewer assay tubes, and the potential for diminished cost were considered advantages of 3-color (vs. 2-color) flow cytometry. In contrast, the increased complexity of factors such as spectral compensation, instrument set up, and data collection/analysis were considered disadvantages. In addition, some antibody combinations are not commercially available, and certain third-color fluorochromes may not be optimal with all flow cytometers. The specifications and recommendations contained herein were developed for use in laboratories that support clinical trials and epidemiologic studies done under the auspices of the National Institute of Allergy and Infectious Diseases, Division of AIDS (NIAID DAIDS). Efforts currently underway by the Centers for Disease Control and Prevention and by the National Committee for Clinical Laboratory Standards (NCCLS) will likely provide additional guidance in this rapidly changing arena. 5.1 3-COLOR IMMUNOPHENOTYPING 5.01. 3-Color Monoclonal Antibody Panel

CCR5 and CXCR4 expression on memory and naive T cells in HIV-1 infection and response to highly active antiretroviral therapy

Objective: To measure CCR5 and CXCR4 chemokine receptor expression on CD4 and CD8 T cells in HIV‐1 infection and to relate levels to the distribution of CD45RO memory and CD45RA‐naive subsets, measures of disease activity, and response to highly active antiretroviral therapy (HAART). Design: Fourteen untreated HIV‐1‐infected patients, 18 patients at 3‐ to 4‐weeks after beginning HAART, and 35 uninfected control subjects were studied. Methods: Four‐color cytofluorometry with appropriate conjugated monoclonal antibodies (mAbs) was performed to define CD45RA and CD45RO subsets of CD4 and CD8 T cells and measure their expression of CCR5, CXCR4, and CD38. Results: HIV‐1‐infected patients had higher CCR5 levels and lower CXCR4 levels on CD4 and CD8 T cells and their CD45RO/CD45RA subsets than control subjects did. However, CCR5 elevation was statistically significant only for CD4 T cells and their subsets, and CXCR4 depression was significant for CD8 T cells and their subsets (and for CD4:CD45RO cells). The elevation of CCR5 and depression of CXCR4 were not due to shifts in CD45RO/CD45RA subset proportions but to upregulation or downregulation within the subsets. CCR5 elevation on CD4 T cells was significantly restored toward normal by HAART, but the CXCR4 depression was not. CCR5 expression but not CXCR4 expression correlated with other measures of immunodeficiency (CD4 T‐cell levels), active infection (viral load), and cellular activation (CD38). Conclusions: CCR5 elevation is a concomitant of immune activation and viral replication that occurs in HIV‐1 infection, but the relation of CXCR4 depression to severity of infection, disease progression, and response to therapy remains undefined.

Adoption of Single-Platform Technologies for Enumeration of Absolute T-Lymphocyte Subsets in Peripheral Blood

The enumeration of specific lymphocyte subsets (flow cytometric immunophenotyping) has become a routine and indispensable procedure in the evaluation, prognosis, and diagnosis of a variety of clinical conditions. Over the past 20 years we have witnessed remarkable changes in the technology of “

Biosafety guidelines for sorting of unfixed cells

The International Society of Analytical Cytology (ISAC) Biohazard Working Group presents guidelines for sorting of unfixed cells, including known biohazardous samples, using jet-in-air, deflected-droplet cell sorters. There is a risk that personnel operating these instruments could become exposed to droplets and aerosols containing biological agents present in the samples. The following guidelines can aid in the prevention of exposures of laboratory personnel to pathogens contained in the sort samples. The document provides biosafety recommendations for sample handling, operator training and protection, laboratory facility design, and instrument setup and maintenance. In addition, it describes in detail methods for assessment of instrument aerosol containment. Recommendations provided here may also help laboratories to obtain institutional and/or regulatory agency approval for sorting of unfixed and known biohazardous samples.

Evaluation of alternative CD4 technologies for the enumeration of CD4 lymphocytes

Enumeration of CD4+ T cells from persons infected with the human immunodeficiency virus (HIV) is important. Traditionally this measurement has been calculated by multiplying the percent of lymphocytes that are CD4+ T cells (from flow cytometry) and the number of lymphocytes per microliter of blood (from hematology measures). Recently, several manufacturers have developed new techniques for determining absolute numbers of CD4+ and CD8+ cells without the need for the flow cytometer and hematology analyzer. We evaluated these methods for accuracy (comparison with traditional methodology) and precision (variability of replicate determinations) as well as for use with specimens older than 1 day. Precision of the assays as determined by the coefficients of variation (CV) from replicates varied from about 3% to about 12%, depending on the assay and the HIV status of the patient. Correlation coefficients of test method results with the standard methodology (r) were all greater than 0.9, and in most cases slopes were close to 1.0 for both CD4 and CD8. Though each methodology will meet different requirements in the laboratory, our results indicate that these assays are all acceptable for enumerating CD4 and CD8 cells in HIV+ as well as HIV- patients.

Enumeration of Antigen Sites on Cells by Flow Cytometry

We evaluated a cytofluorometric method for determining the number of antigens expressed on the cell surface of human lymphocytes. Using beads that have a known number of binding sites for mouse immunoglobulin and monoclonal antibodies specific for various antigens on human lymphocytes, we found that this system is quite reproducible, reliable and technically easy to perform. The greatest source of variation in expression of cell surface antigens is interdonor variability.