Intracellular Staining With True-Phos™ Perm Buffer in Whole Blood v2 (protocols.io.bac6iaze)

ABSTRACT Tests based on the gamma interferon (IFN-γ) assay (IGA) are used as adjunctive tools for the diagnosis of Mycobacterium tuberculosis infection. Here we compared in-house and commercial whole-blood IGAs to identify a suitable assay for the surveillance of tuberculosis in population studies. The IGAs were selected on the basis of the ease with which they are performed and because they require a small amount of a biological sample and do not require cell purification. Since a “gold standard” for latently M. tuberculosis-infected individuals is not available, the sensitivities and the specificities of the IGAs were determined with samples from patients with clinically diagnosed active tuberculosis and in Mycobacterium bovis BCG-unvaccinated healthy controls. The in-house tests consisted of a bulk assay based on diluted whole blood and a single-cell assay based on IFN-γ intracellular staining. The commercial assays used were the QuantiFERON-TB-Gold (Q-TB) and the Q-TB in-tube tests. When the purified protein derivative was used as the antigen, in-house whole-blood intracellular staining was found to be highly discriminatory between active tuberculosis patients and BCG-vaccinated healthy controls, whereas the other IGAs did not discriminate between the two categories of patients. When M. tuberculosis-specific antigens were used, a very strong agreement between the results of the Q-TB in-tube assay and the clinical diagnosis was observed, while the Q-TB assay, performed according to the manufacturer's instructions, showed a significantly lower performance. Intriguingly, when the test was performed with RD1 proteins instead of peptides, its sensitivity was significantly increased. The in-house assay with diluted whole blood showed an elevated sensitivity and an elevated specificity, and the results agreed with the clinical diagnosis. Considering that the in-house assay uses 1/20 of the sample compared with the amount of sample used in the commercial IGA, it appears to be particularly promising for use in pediatric studies. Overall, the different assays showed different performance characteristics that need to be considered for surveillance of tuberculosis in population studies.

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Fine Structure of Platelet Interaction with a New Microcrystalline Collagen Preparation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050305 ◽

1974 ◽

Vol 32 ◽

pp. 58-59

Author(s):

W. H. Zucker ◽

R. G. Mason

Keyword(s):

Fine Structure ◽

Platelet Aggregation ◽

Hydrochloric Acid ◽

Whole Blood ◽

Platelet Adhesion ◽

Hemostatic Agent ◽

Native Collagen ◽

Fibrillar Morphology ◽

Clinical Interest ◽

New Form

Platelet adhesion initiates platelet aggregation and is an important component of the hemostatic process. Since the development of a new form of collagen as a topical hemostatic agent is of both basic and clinical interest, an ultrastructural and hematologic study of the interaction of platelets with the microcrystalline collagen preparation was undertaken.In this study, whole blood anticoagulated with EDTA was used in order to inhibit aggregation and permit study of platelet adhesion to collagen as an isolated event. The microcrystalline collagen was prepared from bovine dermal corium; milling was with sharp blades. The preparation consists of partial hydrochloric acid amine collagen salts and retains much of the fibrillar morphology of native collagen.

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3-D organization of fibrinogen receptors using computer reconstruction and whole-mount microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102237 ◽

1988 ◽

Vol 46 ◽

pp. 30-31

Author(s):

E. T. O'Toole ◽

R. R. Hantgan ◽

J. C. Lewis

Keyword(s):

Whole Blood ◽

Colloidal Gold ◽

Blood Platelets ◽

Cell Contact ◽

Computer Assisted ◽

Adherent Cells ◽

Differential Centrifugation ◽

Computer Reconstruction ◽

Sds Page ◽

Whole Mount

Thrombocytes (TC), the avian equivalent of blood platelets, support hemostasis by aggregating at sites of injury. Studies in our lab suggested that fibrinogen (fib) is a requisite cofactor for TC aggregation but operates by an undefined mechanism. To study the interaction of fib with TC and to identify fib receptors on cells, fib was purified from pigeon plasma, conjugated to colloidal gold and used both to facilitate aggregation and as a receptor probe. Described is the application of computer assisted reconstruction and stereo whole mount microscopy to visualize the 3-D organization of fib receptors at sites of cell contact in TC aggregates and on adherent cells.Pigeon TC were obtained from citrated whole blood by differential centrifugation, washed with Ca++ free Hank's balanced salts containing 0.3% EDTA (pH 6.5) and resuspended in Ca++ free Hank's. Pigeon fib was isolated by precipitation with PEG-1000 and the purity assessed by SDS-PAGE. Fib was conjugated to 25nm colloidal gold by vortexing and the conjugates used as the ligand to identify fib receptors.

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