Immunophenotyping using gold or silver nanoparticle-polystyrene bead conjugates with multiple light scatter.

BACKGROUND: The type of antibody-conjugated polystyrene (PS) latex beads for use as light scatter shift agents for targeted lymphocyte populations in whole blood has been expanded to include gold and silver nanoparticle-aminodextran-PS latex bead conjugates with antibodies. The linkers between antibody and colloidal metal were an aminotrithiol ligand or aminodextran polymer molecules. METHODS: A modified flow instrument, including forward light scatter (FS), side light scatter (SS), light scatter at other intermediate angle ranges, LMALS (10-20 degrees ) and UMALS (20-65 degrees ) was used for simultaneous bead probe measurements. A conventional flow cytometer was used in simultaneous bead-fluorescent marker experiments. RESULTS: Two mutually exclusive cell populations, CD4+ and CD8+ lymphocytes, have been simultaneously enumerated in blood by using a mixture of CD4-PS, CD8-Au-PS or CD4-Au-PS, CD8-PS beads, and one laser line, 633 nm, excitation. Similar measurements were made with mixtures of CD4-PS, CD8-Ag-PS or CD4-Ag-PS, CD8-PS beads. Also, simultaneous use of bead and fluorescent markers mixed with whole blood was demonstrated with CD4-PS beads and with the CD4-RD1/CD8-FITC dual marker. CONCLUSIONS: Enumeration of CD4 and CD8 lymphocytes in whole blood by light scatter parameters only compared well with standard analyses with fluorescent markers. In simultaneous bead-fluorescent marker labeling of lymphocytes, the labeled bead had to be mixed first with cells in whole blood.

Tris(3-mercaptopropyl)-N-glycylaminomethane as a new linker to bridge antibody with metal particles for biological cell separations.

Conjugates of nickel beads with CD8 and anti-red blood cell KC16 antibody were prepared by using the aminotrithiolate "spider" ligand, tris(3-mercaptopropyl)-N-glycylaminomethane, in its new function as a linker between the surface of nickel beads and antibody via activation of spider ligand attached to nickel beads with the common, heterobifunctional cross-linker, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). Raw nickel beads were cleaned by either mild sonication in a bath or by stronger probe sonication to remove surface nickel oxide layers, before attachment of the spider ligand. Scanning electron micrographs of the nickel beads before and after probe sonication showed a marked change from a corrugated to a smooth bead surface. Analyses of the supernatants of conjugation mixtures for antibody gave surface densities of 2.5-5.2 mg/m(2) for CD8 and 0.6-12 mg/m(2) for KC16 antibody runs. The antibody-spider-nickel bead conjugates were used in magnetic bead depletions of targeted CD8+ lymphocytes or red blood cells (rbcs) in whole blood of normal donors. For CD8 cell depletions, the undepleted controls and supernatants of depleted samples were analyzed for CD8/CD4 cell populations by flow cytometry with appropriate fluorescent antibody markers. Enumeration of red blood cells, white blood cells (wbcs), and platelets (plts) in undepleted controls and supernatants of depleted samples were carried out on appropriate hematology counters. Whole blood titer results with various lots of either CD8-spider-nickel or KC16-spider-nickel bead conjugates showed varying degrees of depletion ability as indicated by bead-to-cell ratios of 2-32 for CD8 beads and by rbc-to-bead ratios of 1.2-10 for KC16 beads. Moreover, varying degrees of specificity of CD8 beads for CD8+ cells over CD4+ cells and of KC16 beads for rbcs over white blood cells and platelets were observed from the normalized nontargeted cell population figures in undepleted controls versus supernatants of depleted samples.

Severe diarrhea due to Cokeromyces recurvatus in a bone marrow transplant recipient.

Cokeromyces recurvatus, a sporangiola-forming dimorphic fungus, is a rare cause of urogenital infection in humans. We report here a case of severe watery diarrhea due to C. recurvatus, which was treated successfully with high-dose oral nystatin therapy. We speculate that our patient was probably predisposed to infections due to opportunistic organisms, such as C. recurvatus, because of post-transplantation immunosuppression. To our knowledge, our patient represents the first documented case of diarrhea due to C. recurvatus in man, and this case highlights the potential pathogenic capability of this opportunistic organism in immunosuppressed patients.

A method for the covalent attachment of cells to glass slides for use in immunohistochemical assays.

A method for covalently attaching viable cells to glass slides is described. The cells are linked covalently to the slide without the use of drying or other fixation procedures that normally destroy cellular surface antigens. The attachment is permanent, and the slides with attached cells may be cryopreserved. The use of the avidin-biotin-peroxidase procedure with this procedure shows excellent sensitivity comparable to flow cytometry.

Erythroleukemia in a child: value of immunocytochemistry and transmission electron microscopy in its diagnosis.

A 4 1/2-year-old girl presented with erythroleukemia (EL) that mimicked acute lymphocytic leukemia (ALL). The diagnosis was established with transmission electron microscopy (TEM) and immunocytochemical staining for hemoglobin. Erythroleukemia may present in childhood and may require TEM and immunocytochemistry to make the diagnosis. The correct diagnosis is extremely important since EL has a much poorer prognosis and requires entirely different therapy. Therefore, it is most important to consider EL in those cases of ALL in which the data are not clear-cut and in which the patient fails to respond.

Immunologic characterization of a helper T-cell lymphoma.

The lymphocytes of a patient with a T-cell non-Hodgkin's lymphoma with peripheral blood involvement and polyclonal hypergammaglobulinemia were characterized in terms of surface markers and immunologic functions. Using the fluorescence-activated cell sorter and employing various monoclonal antibodies against T-cell surface antigens, it was shown that almost all of the patient's peripheral blood lymphocytes were positive for OKT4 and 9.3, antibodies that recognize helper T-cell subset. The circulating lymphoma cells had typical characteristics for T cells; they formed spontaneous rosettes with sheep erythrocytes and stained with the pan-T-cell antibodies 9.6 and 10.2, but did not react with other anti-T-cell monoclonal reagents such as OKT3, UCHT-1, and 3A1. The cells appeared to be mature by the fact that they did not stain with OKT6, and terminal deoxynucleotidyl transferase was undetectable. Functionally, they were able to provide "help" for antibody production, and they could be stimulated to produce moderate amounts of interleukin-2, while unable to proliferate in response to mitogens. Morphologically, some of the lymphocytes showed a deeply cleaved nucleus.

Detection of platelet antibody using rosette technique with anti-IgG antibody-coated polyacrylamide gel: a preliminary report.

Paraformaldehyde-fixed platelets from normal donors were used for detection of antibody to platelet by in vitro sensitization (indirect method) utilizing rabbit anti-human IgG heavy chain specific antibody-coated polyacrylamide gels (Immunobeads). The sensitized platelets formed rosettes with Immunobeads and the positive rosette count was over 30%, while control showed less than 8% when normal sera were used. This method was also applicable for detecting antibody-sensitized platelets in vivo (direct method) in patients with autoimmune thrombocytopenia. This method is simple, rapid and reproducible for clinical use. Direct and indirect immunofluorescent antibody tests and a blocking test with anti-human serum also supported the results of Immunobead rosetting technique.

Platelet migration inhibition test for platelet antibodies. Parameters and simplified methodology.

A simplified methodology and parameters associated with the performance of a platelet migration inhibition test for the detection of antiplatelet-associated antibodies is described.