Janet K.A. Nicholson

Use of CD45 fluorescence and side-scatter characteristics for gating lymphocytes when using the whole blood lysis procedure and flow cytometry.

The light-scatter characteristics of lymphocytes are commonly used to gate lymphocytes for further analysis in a lysed whole-blood assay. Because lymphocytes can be identified by antigens that they possess, a light-scatter gate can be validated by measuring parameters other than light scatter. When a specimen possesses poor light scatter (usually from contaminating nonlymphocytes within the light-scatter gate for lymphocytes), the quality of the gate and, thus, the analyses of lymphocyte subsets can be compromised. We present data to demonstrate the use of CD45 fluorescence combined with side scatter (SSC) for analyzing lysed whole-blood specimens. When we compared CD45/SSC to light scatter (forward and side scatter) for validating a lymphocyte gate, both methods performed similarly in recovering as many lymphocytes as possible in the gate (lymphocyte recovery); however, the CD45/SSC gate had fewer contaminants within the gate (lymphocyte purity). Replicate CD3 values from the CD45/SSC gate were less variable than those from the light-scatter gate, confirming that most of the variability in a light-scatter gate is due to nonlymphocyte contaminants in the gate. We propose that lymphocytes be gated using CD45 fluorescence as well as side-scattering properties and that CD3 also be included in each data analysis tube for quality control.

Comparison of T and B cell analyses on fresh and aged blood

We have compared T and B cell analyses using whole blood, separated lymphocytes, and separated, frozen, then thawed lymphocytes to see how long blood can be kept before separation and analysis. We also examined the effect of various anticoagulants and the effect of diluting blood in culture media on T and B cell analysis over time. We found that the whole blood method is a very reliable method for T and B cell analysis, even 4 days after the blood is drawn, provided that heparin or ACD is the anticoagulant used. Separated lymphocytes and cryopreserved lymphocytes from blood that was separated within 24 h of collection was satisfactory; however, results were less consistent if separation was delayed more than 24 h. For lymphocyte separation, blood collected in heparin or ACD held up better over time than did blood collected in EDTA, and dilution with either RPMI 1640 or McCoys medium gave better lymphocyte separation in older blood.

Comparison of monocyte separation methods using flow cytometric analysis

We isolated normal, nonactivated human monocytes from peripheral blood by four different methods: (1) rosetting with sheep erythrocytes pretreated with 2-aminoethylisothiouronium bromide hydrobromide (AET) followed by monoclonal antibody (OKT3 (CD3), B1 (CD19), Leu7, Leu11 (CD16] and complement treatment; (2) adherence to gelatin/plasma-coated flasks; (3) adherence to plastic dishes; and (4) separation by the Sepracell technique. We monitored these monocyte separations by determining cell recoveries, OKT4A+ lymphocyte contamination, monocyte binding to human immunodeficiency virus (HIV), number of non-specific esterase-positive cells, and proportion of mononuclear cells reactive with a battery of monoclonal antibodies specific for monocytes. Our results indicate that of the four methods compared, adherence to gelatin/plasma-coated flasks produced the highest purity, recovery, and satisfactory binding to HIV with the fewest contaminating CD4+ T cells.

Selection of anticoagulants for lymphocyte immunophenotyping: Effect of specimen age on results

In a multi-center study, whole blood specimens from 31 HIV-positive and 43 HIV-negative donors were collected in three different anticoagulants and assayed for lymphocyte subsets fresh (within 6 h), and 1 and 2 days later. Each center prepared the specimens by their routine whole blood lysis procedure, labeling with a recommended panel of two-color monoclonal antibody combinations. 1 day (up to about 30 h) after blood collection, the results obtained from blood collected in EDTA (ethylenediamine tetra-acetate), ACD (acid citrate dextrose), and heparin were similar to fresh. Up to 48 h, only ACD and heparin, not EDTA, yielded results similar to fresh specimens. These results were similar for both HIV-positive and -negative specimens.

Recovery of infectious human immunodeficiency virus from cells treated with 1% paraformaldehyde

Fixation and preservation of cells with 1% paraformaldehyde is a common practice in laboratories performing flow cytometric analysis of cells from persons infected with human immunodeficiency virus (HIV). Previous studies indicated that 1% paraformaldehyde effectively inactivates cell-free HIV. In this study, we tested the inactivation capacity of 1% paraformaldehyde on a cell line chronically infected with HIV and human peripheral blood lymphocytes acutely infected with the virus. We found a significant decrease in the infectious titer of the cells after treatment with 1% paraformaldehyde. However, we did not find total inactivation of the specimens treated for up to 18 h.

Alterations of functional subsets of T helper and T suppressor cell populations in acquired immunodeficiency syndrome (AIDS) and chronic unexplained lymphadenopathy.

We have studied the distribution of Leu8 and Ia on T cells of the helper (T4) and suppressor (T8) phenotype in homosexual men with chronic unexplained lymphadenopathy, homosexual men with the acquired immunodeficiency syndrome (AIDS), and homosexual and heterosexual controls. Decreased numbers of helper T cells in lymphadenopathy patients are due to a decrease in the T4:Leu8+ subset, whereas in AIDS patients, both the T4:Leu8+ and the T4:Leu8− subsets were decreased.

Inactivation of HIV-infected H9 cells in whole blood preparations by lysing/fixing reagents used in flow cytometry

Reagents that lyse red blood cells and fix white blood cells were tested for their ability to inactivate cell-associated human immunodeficiency virus (HIV). Whole blood was spiked with cells from an HIV-positive cell line (H9), lysed, and fixed. The cell preparations were then cocultured with T cell blasts in serial ten-fold dilutions to rescue infectious virus and measure viral titer. All commercial lysing and fixing reagents tested inactivated cell-associated HIV by 3-5 logs, while ammonium chloride had little effect. Although an additional incubation with 1% formaldehyde for 30 min did not increase the effectiveness of the commercial lysing/fixing reagents, it did inactivate cell-associated HIV in blood treated with ammonium chloride.

Phenotypic distribution of T cells of patients who have subsequently developed AIDS

In a previous study of asymptomatic homosexual men, we found that CD8+ T-cell levels were higher in homosexual men infected with the human immunodeficiency virus (HIV) than in uninfected homosexual men because of higher numbers of CD8+ T cells that do not express the Leu15 marker, a phenotype associated with cytotoxic function. Among infected men, there was a positive correlation between the number of CD8+Leu15- T cells and the number of CD4+ T cells. If CD4+ T-cell levels are taken as a measure of severity of HIV infection and immunodeficiency, these results suggested that higher CD8+Leu15- cells may represent a phenotypic profile associated with less severe infection or better control of infection. In the present study, we extend the analysis to include a group of men who progressed to AIDS but were studied well before the onset of AIDS, and we compare results of CD8 subset analyses with results of infected men who have not progressed to AIDS. Phenotypic subsets associated with helper, suppressor, cytotoxic, and natural killer cell function were determined by two-color immunofluorescence. The only phenotypic subset that distinguished men who progressed to AIDS from those who have not was lower numbers of CD4+ T cells in the former group. If CD8+Leu15- cell numbers (or other phenotypic subsets examined) reflect effective control of HIV infection, the relationship is not strong enough to be of prognostic or predictive value with respect to outcome of infection.

Lymphocyte immunophenotyping at the Centers for Disease Control: The program and special studies☆

Lymphocyte immunophenotyping has been done at the Centers for Disease Control since 1981. The technology is constantly evolving, and changes have been instituted to keep up with and improve this technology. In the last 7 years we have analyzed approximately 8300 specimens from patients and persons at risk in projects related to the acquired immunodeficiency syndrome. Though several subsets of lymphocytes have been found to be increased or decreased in HIV infection, no lymphocyte determination is more sensitive than CD4 alone in predicting the outcome of human immunodeficiency virus infection.

Inhibition of adenosine deaminase leads to enhanced antibody responses in the mouse

Inhibition of adenosine deaminase activity leads to decreased cellular immunity. The effect of deoxycoformycin (DCF), a potent inhibitor of adenosine deaminase, on the ability of mouse spleen cells to generate antibody responses in vitro has been examined. With either continuous exposure to or pretreatment of the cells with deoxycoformycin, there was a decrease in cell survival and an increase in antibody-producing cells in the surviving cell population. To identify the cell population most susceptible to the inhibitor, the spleen was separated into B-cell, and T-cell, and macrophage components and each population was pretreated with deoxycoformycin before combination with its complementary treated or untreated population. Deoxycoformycin pretreatment had no effect on macrophages or B cells; however, pretreatment of the T cells resulted in increased antibody responses. When T cells and B cells were both pretreated and combined, there was a synergistic increase in the antibody response. In addition, supernatants from cultures in which both B cells and T cells had been pretreated with DCF were capable of enhancing antibody responses in cultures containing DCF-treated T cells. Though adenosine was increased in the stimulatory culture supernatants, adenosine alone did not enhance antibody responses in either untreated or DCF-treated cultures.