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

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 unmask-

Pharmacodynamic monitoring of BAY 43‐9006 (Sorafenib) in phase I clinical trials involving solid tumor and AML/MDS patients, using flow cytometry to monitor activation of the ERK pathway in peripheral blood cells

“…The whole blood fixation/density gradient separation protocol used in the MDS/AML trial is technically de- manding and laborious, and was recently replaced in our laboratory by a whole blood fixation/red cell lysis technique (16). As well as being technically simpler, this new technique gives much better preservation of surface markers and light scatter than was obtained in the studies reported in the present article.…”

“…6). Note that these experiments were done using a later modification of the whole blood fixation protocol (16), which accounts for the greater levels of pERK following PMA activation compared to the AML/MDS trial data shown in Figure 5.…”

Validation of a multiparameter flow cytometry method for the determination of phosphorylated extracellular‐signal‐regulated kinase and DNA in circulating tumor cells

“…Fixation is necessary because the time interval and manipulations used to eliminate blood cells have the potential to introduce artifacts (25), as pERK is part of a signaling network that responds to the environment and turns over rapidly.…”

A phase I/II study of imatinib plus reinduction therapy for c-kit-positive relapsed/refractory acute myeloid leukemia: inhibition of Akt activation correlates with complete response

“…Flow cytometry was used to measure the pharmacodynamic effects of imatinib treatment on signaling via the c-kit receptor in peripheral blood blast cells. This involved acute ex vivo activation by the c-kit ligand SCF, as previously reported, 19,20 using a whole-blood assay procedure developed by Chow et al 21 Based on preliminary dose/response and kinetic studies, a final SCF concentration of 50 ng/ml was added to 100 ml aliquots of undiluted whole blood at 371C, followed by fixation after 5 min with the addition of 10% methanol-free formaldehyde to give a final concentration of 4%. After 10 min, Triton X-100 was added to give a final concentration of 0.1% for 30 min to lyse red blood cells.…”

“…Samples were then either held in a 20% glycerol/10% fetal calf serum freezing mixture at À701C or processed immediately for flow cytometry. Samples were washed, treated with 50% methanol as previously described 21 and then resuspended at 10 6 cells in 50 ml. Cells were dual stained for phosphorylated ERK (pERK) using an Alexa-488-conjugated mouse monoclonal antibody and phosphorylated Akt (pAkt) using an Alexa-647-conjugated rabbit monoclonal antibody (both from Cell Signaling Technology, Danvers, MA, USA), which measures the serine 473 phosphorylation site of Akt.…”