Methods and means for monitoring disruption of tissue homeostasis in the total body

A method for determining the health status of a subject, for early detection of tissue damage, for early diagnosis and monitoring of a disease, and/or for evaluation of treatment effectiveness in a subject using circulating tissue macrophages as a mirror of disrupted tissue homeostasis and disease, the method comprising the steps of:

• a) providing a peripheral blood sample from the subject which contains circulating tissue macrophages (CTM);
• b) staining said CTM with a panel of differentially-labeled distinct antibodies against the backbone markers CD14, CD16 and CD300e for the identification and enumeration of different CTM subsets;
• c) fixation, permeabilization and staining of the CTM using one or more detecting antibodies directed against one or more epitopes on at least one protease-induced protein fragments derived from intracellular degradation of a non-CTM protein by individual CTM in their tissues of origin, thereby identifying at least one subset of circulating tissue-specific macrophages (CTSM);
• d) multiparameter flow-cytometric analysis of said stained CTM and CTSM by gating for the backbone markers CD14, CD16 and CD300e to determine the amount of signals of each distinct labeled antibody associated with individual cells, wherein the analysis comprises performing a gating strategy based on the cell surface expression of CD300e, CD14 and CD16, in combination with side scatter (SSC) analysis, and wherein the gating strategy is composed of (i) an inclusion step to include both classical monocytes and CTMs based on CD300e expression in combination with side scatter (SSC) analysis, and (ii) a subsequent subset identification step to discriminate the classical CD14high/CD16−;
• e) determining the relative and absolute number of individual cells within each CTM subset and each specific subset of CTSM that express each of the measured intracellular epitopes;
• f) calculating (i) the relative and absolute number of cells within each CTM subset and each specific subset of CTSM which each originate from different normal and altered tissues as defined by a set of individual protease-induced protein fragments evaluated, and ii) the amount of antibody-related signal associated to every individual intracellular peptide evaluated to obtain a test CTSM staining profile, and;
• g) comparing the test CTSM staining profile with a normal CTSM staining profile for each tissue evaluated, wherein an aberrant test staining profile is indicative of tissue damage, an altered tissue homeostasis, the presence of a disease, and/or treatment effectiveness versus resistance.