Whole blood preparation for cytometric analysis of cell signaling pathways

A method of preparing a biological sample containing red blood cells and white blood cells for measurement of protein epitopes, said method allowing for the preservation of intracellular protein epitopes for detection, said method comprising the steps of:

• (a) a fixation step comprising contacting a biological sample containing red blood cells and white blood cells with a fixative, wherein said fixative is added in an amount to achieve a final concentration sufficient to crosslink proteins, lipoproteins and nucleic acid molecules;
• (b) a detergent step comprising contacting said sample from step (a) with a detergent, wherein said detergent is added in an amount to achieve a final concentration sufficient to lyse said red blood cells and permeabilize said white blood cells;
• (c) a detergent removal step comprising removing said detergent from said sample;
• (d) an alcohol step comprising contacting said sample with an alcohol in an amount to achieve a final concentration, which is sufficient to unmask said intracellular epitopes that have been made inaccessible by the fixative without reducing the reactivity of cell surface epitopes, wherein the alcohol step is performed after the detergent step, after the detergent removal step, or together with the detergent step;
• and,
• (e) a labeling step comprising contacting said sample with a detectable binding agent specific for one or more protein epitopes after the detergent removal step and the alcohol step.