Abstract Purpose: Clinical assessment of quantitative fluorescein angiograms based on novel instrumentation adaptation. Methods: Fluorescein angiograms were simultaneously performed on patient’s retinas and on standard cells composed of two sets of five standard fluorescein solutions mimicking the environmental conditions of the plasma and vitreous, respectively. Regular gray‐scale fluorescein angiograms were converted into absolute concentration image maps, after a deconvolution step for the separation of plasma and vitreous fluorescence contributions, taking into account the respective fluorescein quantum efficiencies. Diabetic retinopathy eyes, eyes before and 3 days after laser photocoagulation, and fellow‐eyes from patients with choroidal neovascularization were imaged using this new technique. Results: Repetitive measurements over patients clearly show the advantage of correcting for instrumentation setup. Comparison of scans taken before and after laser photocoagulation demonstrates background values within the same range and the expected increase over laser spots. Conclusions: Quantitative fluorescein angiograms compare favorably with conventional fluorescein angiograms by removing instrumentation setup bias and by allowing absolute concentration image maps to be built and comparable. Furthermore, the use of a non‐linear color‐coded map allows the detection of subtle differences not visible on regular fluorescein angiograms. It is realized that this first step needs to be complemented by other standardization procedures, e.g., by fundus reflectance.
A procedure is presented to convert the comparison of measured fluorescence signals into a comparison of fluorescence yields (FY). The fluorescence yield, which is a property of a solution or a suspension, is defined as the product of the fluorophore concentration and the molecular quantum yield. The paper revises the measurement model which relates the measured fluorescence signal to the FY. The equality of FY of two solutions provides an equivalence between the concentrations of fluorophore in the two solutions. The equivalence is the basis for quantitation in terms of molecules of equivalent soluble fluorophore (MESF). The quantitation procedure starts with the measurement of fluorescence signals from a serial dilution of fluorescein solutions to obtain a calibration of a fluorometer. The fluorometer is used to measure the fluorescence signal of a suspension of microspheres with immobilized fluorescein isothiocyanate (FITC). The calibration is used to obtain the concentration of soluble fluorophores which gives the same fluorescence signal as the microsphere suspension. The number concentration of microspheres is measured and the equality of fluorescence yields is used to obtain the number of soluble fluorescein molecules equivalent to a single microsphere.
The flow cytometric analysis of cells from patients with known or suspected hematological malignancy encompasses issues of specimen triage (is the test necessary?),technology (actual laboratory performance of the test), medical interpretation (analysis of technical data in the medical context of the patient), and reporting of the results in a manner meaningful to the treating physician.For the purpose of this report, the technical aspects of flow cytometric immunophenotyping are defined by the following components: (1) acquisition of the patient specimen and transport to the laboratory, (2) sample preparation and staining, (3) instrument quality control and quality assurance, and (4) data acquisition on the flow cytometer and data storage.Unlike the more esoteric aspects of the analysis and interpretation of immunophenotypic patterns in hematological malignancy, there is some consensus opinion established regarding many of the technical aspects common to this and other applications of clinical flow cytometry.Previous consensus proceedings have addressed, particularly, issues of instrumentation and specimen handling as they relate to analysis of leukemia, lymphoma (21,59) and lymphocyte subset analysis for monitoring patients with HIV infection (11,44,45,52).This report also reiterates important points made in the previous work and elaborates on issues specific to the flow cytometric analysis of hematopoietic malignancies not previously discussed.It should be understood that the recommendations of this conference are presented as a basis for further discussion and enhancement.It also should be noted that the recommendations presented by this committee strongly reflect discussion and recommendations by the other core committees involved in the formulation of this consensus regarding the utility of flow cytometry for the analysis of hematological malignancies.This is particularly true for issues such as antibody panel selection and validation and importance of fluorescence standardization and quantification, as well as a number of other topics that reflect upon the technical aspects of the test.
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