Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations

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Abstract
Background Previous studies of intracellular expression of phospho-epitopes in human leukocytes using flow cytometry have used erythrocyte removal or lysis before fixation. Because many of the phospho-epitopes of interest are part of signaling networks that respond to the environment and turn over rapidly, the interval and manipulations used to eliminate erythrocytes from samples have the potential to introduce artifacts. We report a procedure to fix samples containing red blood cells with formaldehyde and then remove erythrocytes by lysis. Detection of phospho-Thr 202/Tyr 204-p44/42 extracellular-regulated kinase (ERK) after phorbol ester acetate (PMA) stimulation was used as a model to measure phospho-epitopes in leukocyte populations in whole blood. Methods Normal blood samples were activated with PMA followed by formaldehyde fixation and subsequent treatments with detergents and protein denaturants. The effects of each treatment were monitored by light scatter, selected CD expression intensity, and phosphorylated ERK (pERK) expression. Results Red cells could be lysed using 0.1% Triton X-100 after brief fixation of whole blood with 2% or 4% formaldehyde. Light scatter improved as a function of formaldehyde concentration and inversely with MeOH concentration. CD3 signal intensity increased when MeOH concentration was reduced. The ratio of pERK immunofluorescence in PMA-stimulated versus nonstimulated (control) samples was highest with high MeOH (90%) and lowest without MeOH treatment. This pattern is consistent with epitope unmasking by alcohol. The pERK epitope could also be unmasked by treatment with high salt, urea, acid, or heat, but none of these produced the level of unmasking of MeOH and each of these was associated with degradation of light scatter and CD3 staining intensity. The final procedure employed 4% formaldehyde, 0.1% Triton X-100, followed by 50% methanol denaturation. Samples prepared in this way demonstrated good preservation of light scatter and surface immunophenotypic patterns, similar to those obtained using a commercial whole blood/red blood cell lysing system (Q-Prep) and an acceptable PMA-stimulated pERK signal (essentially 100% of CD3+ cells that are pERK positive). Conclusions Brief fixation of whole blood in 4% formaldehyde followed by treatment with Triton X-100 results in erythrocyte lysis and leukocyte light scatter and immunophenotypic features equivalent to those of other commercial lysis reagents. Intracellular pERK staining is significantly improved by treatment with methanol, but levels of MeOH above 50% degrade light scatter and CD3 expression. This protocol (formaldehyde/Triton X-100/MeOH) circumvents potential artifactual changes in phospho-epitopes due to removal of erythrocytes or erythrocyte lysis followed by fixation, and results in a pERK signal that resolves positive from negative cell populations.