Reproducibility of immunological tests used to assess multiple chemical sensitivity syndrome.

Whether persons with multiple chemical sensitivity syndrome (MCS) have immunological abnormalities is unknown. To assess the reliability of selected immunological tests that have been hypothesized to be associated with MCS, replicate blood samples from 19 healthy volunteers, 15 persons diagnosed with MCS, and 11 persons diagnosed with autoimmune disease were analyzed in five laboratories for expression of four T-cell surface activation markers (CD25, CD26, CD38, and HLA-DR) and in four laboratories for autoantibodies (to smooth muscle, thyroid antigens, and myelin). For T-cell activation markers, the intralaboratory reproducibility was very good, with 90% of the replicates analyzed in the same laboratory differing by < or = 3%.

Need for an external proficiency testing program for cytokines, chemokines, and plasma markers of immune activation.

An external evaluation program for measuring the performance of laboratories testing for cytokines and immune activation markers in biological fluids was developed. Cytokines, chemokines, soluble cytokine receptors, and other soluble markers of immune activation (CSM) were measured in plasma from a healthy human immunodeficiency virus (HIV)-seronegative reference population and from HIV-seropositive individuals as well as in supernatant fluids from in vitro-stimulated human immune cells. The 14 components measured were tumor necrosis factor (TNF) alpha, gamma interferon, interleukin-1 (IL-1), IL-2, IL-4, IL-6, IL-10, Rantes, MIP-Ia, MIP-Ibeta, soluble TNF receptor II, soluble IL-2 receptor alpha, beta(2)-microglobulin, and neopterin.

Inter-laboratory relative fluorescence intensity measurements using FlowCal 575 calibration beads: a baseline study.

Twenty-one laboratories participated in a baseline study of their ability to agree on the measurement of fluorescence intensities of a stable multi-peak reference material (FlowCal 575) and of stained and fixed CD4 lymphocytes. Relative fluorescence intensities were calculated as ratios to the most fluorescent bead. The good correlation between laboratories suggests that this simple approach may be useful in multi-laboratory studies.

Assessment of the effects of instrumentation, monoclonal antibody, and fluorochrome on flow cytometric immunophenotyping: a report based on 2 years of the NIAID DAIDS flow cytometry quality assessment program.

This study of the effect on CD4%, CD8%, CD3+8+%, and CD3% of flow cytometer, monoclonal antibody, and fluorochrome was based on 71 whole-blood samples, each evaluated by 42 to 59 laboratories during 2 years of a flow cytometry quality assessment program. For the 24 HIV-positive specimens, FACScans produced significantly lower CD4% values than EPICS-Cs or EPICS Profiles, and for the 47 HIV-negative specimens, FITC was associated with significantly lower CD4% values than PE or RD1, but differences were never larger than 2% and regressions accounted for only 3-12% of the variability. The labs using the most common CD4 technique had significantly higher between-laboratory variability than all other labs grouped together.

Flow cytometric analysis of whole blood lysis, three anticoagulants, and five cell preparations.

We studied the effects of anticoagulants and cell preparation methods on lymphocyte forward-angle scatter (FSC), autofluorescence, and immunofluorescent staining for CD45, CD14, and CD13. Blood samples collected in ethylenediaminetetracetic acid (EDTA), heparin, and acid citrate dextrose (ACD) were processed by using conventional Hypaque-Ficoll (HF) separation and four whole blood (WB) lysis techniques: Immuno-lyse, Q-Prep, FACS Lyse, and Gen Trak Lysis. Lymphocytes prepared by using three of the four whole blood methods gave FCS values comparable to those isolated by HF, while one method (FACS Lyse) gave consistently lower values.

Quality control in the flow cytometric measurement of T-lymphocyte subsets: the multicenter AIDS cohort study experience. The Multicenter AIDS Cohort Study Group.

Since 1984, the Multicenter AIDS Cohort Study (MACS) has utilized four flow cytometry laboratories to measure T-lymphocyte subset levels semiannually in a large cohort of homosexual men. This report summarizes the steps taken in the MACS laboratories to attain comparability of lymphocyte subset determinations across the centers and over time. Identical flow cytometers, monoclonal antibodies, and analytic procedures have been used, and over a period of time, the procedure for sample preparation was also standardized.

Department of Army lymphocyte immunophenotyping quality assurance program.

With the emergence of the human immunodeficiency virus (HIV) epidemic, lymphocyte immunophenotyping has become the single most important laboratory test for clinical management of HIV-infected subjects. To meet this challenge, the department of Army instituted a multicenter lymphocyte immunophenotyping quality assurance (QA) program in March 1986. An integral part of the QA program has been the development of a monthly proficiency testing program to survey the degree of precision and reproducibility of lymphocyte subset determinations within the Army.

Fluorescence microscopic and flow cytometric analysis of bone marrow-derived cells in human epidermis: a search for the human analogue of the murine dendritic Thy-1+ epidermal cell.

Lymphoid cells with an affinity for the epidermis (epidermotropic lymphocytes) have been proposed to play a role in the immune functions of the epidermis. However, antigen-presenting Langerhans cells (LC) and indeterminate cells are presently the only cells in the human epidermis which have been demonstrated to originate in the bone marrow. Recent studies of murine epidermis have identified a population of bone marrow-derived cells which express Thy-1 antigen and which are present in a similar density to, but distinct from, LC.

Detection and enumeration of monocytes in human blood with peanut agglutinin.

Binding of peanut agglutinin (PNA) to normal human peripheral blood mononuclear cells was analyzed on a cell sorter, and compared to the binding of the monocyte specific monoclonal antibodies Mac-1 and Leu-M3. Each of the reagents labeled 9-11% of the mononuclear cells and similar binding patterns were observed. Of the PNA+ cells, 67% adhered to plastic petri dishes, whereas 76% of Mac-1+ cells were adherent.

Segmental homology between T-cell receptors and immunoglobulin variable regions: evidence that antisera to synthetic JH1 peptide react with murine and human T-cell products.

To determine precisely the nature of serological determinants shared between T-cell surface molecules and immunoglobulin variable regions, the capacity of antisera directed against a synthetic peptide corresponding to the entire JH 1 region of classical immunoglobulin plus five residues of the D region were tested for their capacity to bind to T-cell membranes and isolated T-cell products. The anti-JH 1 antisera reacted with normal and monoclonal in vitro grown T-cell lines as judged by microhemagglutination and binding in enzyme-linked immunosorbent assays. Immunologically cross-reactive membrane components disclosed by immunoblot transfer analysis ("Western blots") consisted of major components in the molecular weight range 30-35,000 and minor components in the range 65-70,000.

Interaction of pokeweed mitogen with monocytes in the activation of human lymphocytes.

The present study examines the role of monocytes in the activation of human T cells and B cells by pokeweed mitogen (PWM). The T cell-dependent PWM-induced B-cell activation process was found to be monocyte dependent. Fluorescence-activated cell sorter (FACS) analysis revealed that upon addition to peripheral blood mononuclear cells, fluoresceinated PWM, at concentrations that provided optimal B-cell and T-cell activation, bound predominantly to human monocytes.

The relationship between phospholipid methylation and calcium influx in murine lymphocytes stimulated with native and modified Con A.

Native Con A and two chemical derivatives, divalent dimeric Con A and monovalent dimeric Con A. induced a transient increase of phospholipid methylation, Ca2+ influx, and also increased DNA synthesis in murine lymphocytes. For each of the individual mitogens, the dose-response curves for these three activities were very similar.

Differential binding of fluorescein-labeled lectins to mouse thymocytes: subsets revealed by flow microfluorometry.

Fluorescein-labeled lectins bound to mouse thymocytes were analyzed by flow microfluorometry. This technique has identified several lectins that bind differentially to thymocyte subsets. The most complex fluorescence distributions were obtained using lectins with nominal specificities for galactose or N-acetylglucosamine.

Phospholipid methylation: a biochemical signal modulating lymphocyte mitogenesis.

Phospholipid methylation in murine T lymphocytes but not B cells was stimulated by mitogenic lectins such as concanavalin A and phytohemagglutinin, and the methylation was then returned to the control level by the concomitant activation of phospholipase A2. A parallelism between dose-response curves of concanavalin A for phospholipid methylation and thymidine incorporation was found. Inhibition of either synthesis or degradation of methylated phospholipids resulted in a decrease in the thymidine incorporation.

Comparison of bovine and mouse pituitary glycoprotein hormone pre-alpha subunits synthesized in vitro.

Poly(A)-containing RNA isolated from bovine and mouse pituitaries and a mouse pituitary thyrotropic tumor was translated in a wheat germ cell-free biosynthetic system. A precursor of the glycoprotein hormone alpha subunit, "pre-alpha," was immunoprecipitated from the translation mixtures with antiserum against bovine luteinizing hormone (LH; lutropin) alpha. The specificity of the immunoprecipitation was shown by competition with authentic bovine LHalpha and lack of competition with bovine thyroid-stimulating hormone (TSH; thyrotropin) beta.