Novel immunocytostaining methods for enhanced dye ratio discrimination in rare event detection

A method of preparing a sample containing potential cells of interest, for use in a laser scanning system designed to detect imaging events which contain the potential cells of interest, the method comprising:

• placing a first material containing the potential cells of interest, on a slide;
• associating a first marker or tag to a first type of antibody;
• associating the first type of antibody to a potential cell of interest;
• and
• associating a second marker or tag to a second type of antibody, the second type of antibody configured to target the first type of antibody,
• wherein the first marker or tag and second marker or tag are associated with the respective first type of antibody and second type of antibody in a sequential non-competitive manner.

A method of preparing a sample containing potential cells of interest, for use in a laser based scanning system designed to detect imaging events which contain the potential cells of interest, the method comprising:

• placing a first material containing the potential cells of interest on a slide;
• associating a first type of antibody to a potential cell of interest;
• associating an enzyme to a second type of antibody, the second type of antibody configured to target the first type of antibody;
• adding a reagent containing a component and a first marker or tag, to the second type of antibody, wherein the component is in a non-reactive state;
• altering the component in the non-reactive state to a reactive state;
• and
• depositing a plurality of the components, in the reactive state in proximity to the second antibody, the plurality of components associated with the first marker or tag, wherein the amount of the first marker or tag in proximity to the second antibody are amplified.

A method of preparing a sample containing potential cells of interest, for use in a laser scanning system designed to detect imaging events which contain the potential cells of interest, the method comprising:

• placing a first material, containing the potential cells of interest, on a slide;
• adding a tandem marker or tag which excites at a first frequency and at a second frequency;
• collecting emissions at the first frequency and at the second frequency;
• determining a ratio representing the ratio of the emission characteristics of the tandem marker;
• and
• performing a filtering operation of imaging events, containing the potential cells of interest, using at least the determined ratio to identify false positives of the acquired imaging events.

A laser scanning system for scanning a sample containing potential cells of interest, the scanning system including:

• an imager stage supporting a slide on which the sample is disposed;
• a light source that emits excitation light at the excitation light wavelength, and optics that convert the emitted fluorescent light into collimated excitation light that impinges on the sample, wherein interaction between the excitation light and the sample generates luminescence light at a different wavelength from the excitation light wavelength;
• an input aperture arranged to collect at least a portion of the luminescence light, and
• an output aperture optically communicating with the input aperture and emitting the luminescence light collected by the input aperture,
• wherein the sample is prepared to emit signals having a spectral resolution sufficient to differentiate between a false positive and a true positive for an acquired imaging event, the true positive confirming detection of a cell of interest.