A method of analyzing a sample containing circulating tumor cells (CTCs) using an automated instrument, comprising: depositing a sample from a subject with prostate cancer on a substrate configured for use with the automated instrument, fixing the sample with 0.4% formalin for 1 to 5 minutes followed by 10% neutral buffered formalin for 5 to 15 minutes and methanol for 1 to 5 minutes, permeabilizing the sample with 0.2% Tween 20 in phosphate buffered saline, retaining at least 90% of the CTCs in the sample on the substrate following the fixing and permeabilizing, placing the substrate within the automated instrument, retrieving protein and nucleic acid molecule targets on the sample, wherein the retrieving is done by the automated instrument, wherein retrieving comprises: (i) contacting the sample with CTC identification reagents, wherein the contacting the sample with CTC identification reagents is done by the automated instrument, (ii) contacting the sample with nucleic acid probes directed to ETS related gene, phosphatase and tensin homolog (PTEN), and centromere 10 (CEN-10), wherein the contacting the sample with the nucleic acid probes is done by the automated instrument, imaging the sample, (iii) locating the CTCs in the sample on the substrate by locating the CTC identification reagents, wherein the CTC identification reagents are immunohistochemical reagents directed to a CD45 protein, a cytokeratin protein, an ERG protein, an androgen receptor, a PSMA protein, or combinations thereof, wherein the locating the immunohistochemical reagents is done by the automated instrument, and (iv) spectral imaging of the CTCs by location to detect the nucleic acid probes, wherein the spectral imaging of the CTCs is done by the automated instrument, and analyzing the sample by analyzing the spectral imaging and the detected CTC characterization reagents. depositing a sample from a subject with prostate cancer on a substrate configured for use with the automated instrument, fixing the sample with 0.4% formalin for 1 to 5 minutes followed by 10% neutral buffered formalin for 5 to 15 minutes and methanol for 1 to 5 minutes, permeabilizing the sample with 0.2% Tween 20 in phosphate buffered saline, retaining at least 90% of the CTCs in the sample on the substrate following the fixing and permeabilizing, placing the substrate within the automated instrument, retrieving protein and nucleic acid molecule targets on the sample, wherein the retrieving is done by the automated instrument, wherein retrieving comprises: (i) contacting the sample with CTC identification reagents, wherein the contacting the sample with CTC identification reagents is done by the automated instrument, (ii) contacting the sample with nucleic acid probes directed to ETS related gene, phosphatase and tensin homolog (PTEN), and centromere 10 (CEN-10), wherein the contacting the sample with the nucleic acid probes is done by the automated instrument, imaging the sample, (iii) locating the CTCs in the sample on the substrate by locating the CTC identification reagents, wherein the CTC identification reagents are immunohistochemical reagents directed to a CD45 protein, a cytokeratin protein, an ERG protein, an androgen receptor, a PSMA protein, or combinations thereof, wherein the locating the immunohistochemical reagents is done by the automated instrument, and (iv) spectral imaging of the CTCs by location to detect the nucleic acid probes, wherein the spectral imaging of the CTCs is done by the automated instrument, and (i) contacting the sample with CTC identification reagents, wherein the contacting the sample with CTC identification reagents is done by the automated instrument, (ii) contacting the sample with nucleic acid probes directed to ETS related gene, phosphatase and tensin homolog (PTEN), and centromere 10 (CEN-10), wherein the contacting the sample with the nucleic acid probes is done by the automated instrument, imaging the sample, (iii) locating the CTCs in the sample on the substrate by locating the CTC identification reagents, wherein the CTC identification reagents are immunohistochemical reagents directed to a CD45 protein, a cytokeratin protein, an ERG protein, an androgen receptor, a PSMA protein, or combinations thereof, wherein the locating the immunohistochemical reagents is done by the automated instrument, and (iv) spectral imaging of the CTCs by location to detect the nucleic acid probes, wherein the spectral imaging of the CTCs is done by the automated instrument, and analyzing the sample by analyzing the spectral imaging and the detected CTC characterization reagents.