Cytometry is a well-developed technology for the characterization of hematologic malignancies based on correlated measurement of multiple surface immunophenotypic markers and, less frequently, internal lineage related antigens. Most of these antigens are long-lived with low turnover and are relatively unaffected by sample preparation. Thus, different anticoagulants, overnight storage and shipment, whole blood staining, and several erythrocyte lysis techniques result in reproducible, convenient, and flexible assays. Recently, interest has been stimulated in potentially less robust assays of cell signaling components based on the relatively recent availability of phosphorylation state-specific antibodies (1–6). Because of the quantitative and correlative natures of cytometry, this represents a novel and potentially powerful approach to classification of hematologic malignancies including the selection of patients for molecular targeted therapies and monitoring drug effects in individual patients (1, 6–8).
Analysis of signal transduction pathways by flow cytometry presents technical problems that are not currently encountered in routine clinical applications (1–5). The phosphorylation states of individual signaling elements change rapidly in response to stimuli and therefore may be subject to changes due to sample collection, storage, preparation, and staining that may obscure the signaling state of the condition under study. In a previous publication (1) we used flow cytometry to detect stimulated changes in phosphorylated extracellular-regulated kinase (pERK) that were correlated with western blot analysis. This measurement has been viewed as a surrogate marker for signals originating upstream and traveling through ERK, as an integrating node in studies of drugs that target upstream signaling pathways. However, because the original protocol employed hypotonic lysis before fixation, which could potentially underestimate pharmacodynamic effects due to drug dissociation and reversal of the effect on signaling before fixation, we explored the possibility of immediate fixation followed by erythrocyte lysis or removal.
A second issue with the original procedure was loss of light scatter information due the second step, treatment with 90% MeOH, which was necessary to unmask the pERK epitope. Each of these problems was addressed by increasing the formaldehyde concentration and decreasing the MeOH concentration. We report an improved technique that employs immediate formaldehyde fixation to stop cellular processes followed by erythrocyte lysis followed by denaturation with MeOH. This procedure results in a detectable and acceptable pERK signal, preserves light scatter better than the original protocol, and results in better preservation of cell surface epitopes. Although we report results for ERK phosphorylation, we have since detected robust and specific signals for phospho-S473-AKT, phospho-S235/236-S6 ribosomal protein, and pERK simultaneously by using this protocol (manuscript in preparation). Therefore, we believe that this procedure will be more widely applicable to the study of phospho-epitope expression in whole blood samples.
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- MATERIALS AND METHODS
- LITERATURE CITED
The purpose of this study was to develop a “fix-lyse” method to measure intracellular phospho-epitopes in samples of whole blood, bone marrow, or other fluids containing RBCs. Previous studies by our laboratories (1, 5) and by Krutzik et al. (3, 4) have demonstrated the utility of using brief formaldehyde fixation followed by alcohol for tissue culture cells, human peripheral blood mononuclear cells, or mouse splenocytes. In these studies, samples containing RBCs were first depleted of erythrocytes by lysis. Two problems were evident. First, removal of interfering RBCs (density gradient separation or RBC lysis) allows time and environments during which artifactual changes in phosphorylation may occur. Second, detection of some phospho-specific epitopes (e.g., pERK, pSTAT1, pSTAT5) was significantly improved by protein denaturation with alcohol (3, 4), but alcohol fixation resulted in degradation of the light scatter, a property that has been noted previously. Further, some surface makers, important for subset analysis of leukocytes, were compromised.
To address the first problem, we examined the impact of initial fixation of whole blood samples with formaldehyde followed by detergent treatment on RBC lysis. Fixation of whole blood was initiated to provide the least amount of time between cessation of experimental treatments and fixation of phospho-epitopes in the sample. There have been several studies aimed at whole blood fixation and permeabilization in which light scatter was considered important. One of these is a study by Francis and Connelly (18) describing the properties of the commercial fix and permeabilizing reagent, PermeaFix (Ortho Diagnostic Systems, Raritan, NJ, USA). This single-step reagent produced light scatter resolution of lymphocytes and monocytes equivalent to ammonium chloride lysed samples of whole blood. In addition, leukocyte surface staining was preserved, cells were permeable, and intracellular antigens could be stained. This reagent is now sold by InVirion (Frankfort, MI, USA). We tested this reagent for pERK staining and, like other methods that leave proteins in a native state, the pERK signal was low (Chow and Hedley, unpublished data). Macey et al. (19) tested several other commercial fixation/permeabilization/lysis reagents for whole blood for the ability to preserve light scatter. Results were variable; all procedures affected light scatter but several produced good patterns in which the three main leukocyte subpopulations could be resolved. Neither of these studies indicated what level of formaldehyde was in these commercial reagents. In the present study, we have shown that we can fix whole blood with high concentrations of formaldehyde (2–4%) for brief periods (10 min), and that this will result in preparations in which the erythrocytes can be completely lysed by incubation in non-ionic detergent solutions at low concentration. Although we tested several different detergents, we did not observe an improved effect when compared with Triton X-100. Thus, we have a method in which blood can be treated with a known amount of formaldehyde and RBCs can be lysed after fixation, thus eliminating leukocyte purification steps. In addition, we have demonstrated that this formaldehyde fixation/detergent permeabilization technique, a fix-lyse procedure, maintains light scatter properties of WBC populations sufficient to allow discrimination of all three leukocyte groups and preserve expression of several common leukocyte subset surface markers.
To address the loss of pERK signal, we tested other means of protein denaturation. Among these were high salt, urea, heat, and acid. Although each of these treatments increased the level of pERK detection, none of these produced cell preparations in which the pERK signal was as high as that achieved with 90% MeOH. Previously, it had been shown that light scatter signals of leukocytes could be retained at lower concentrations of alcohol fixation (20). Therefore, we examined this and confirmed and demonstrated that the loss of light scatter, CD3 intensity, and pERK intensity were dependent on alcohol concentration. By lowering the alcohol concentration to 50%, we added a denaturation step and arrived at a whole blood fixation procedure that provided good light scatter patterns and high CD3 intensity and a significantly unmasked pERK signal. Although our original lyse-fix technique (Method A) resulted in a higher pERK signal, poor resolution of WBC populations, and low CD3 intensity, this new approach resulted in an intermediate pERK signal, good resolution of WBC populations, and high CD3 intensity. Because unmasking is likely to be the result of protein denaturation and extraction, this intermediate pERK signal may be the result of increased retention of molecules that could be extracted by the alcohol step, but are retained by the additional cross-linking at higher formaldehyde concentration. For pERK and other highly expressed epitopes, this is not a severe limitation. The fraction of pERK-positive cells remains the same. Detecting less than the total number of epitopes in the cells becomes important only when the total number of epitopes is low to begin with, or in the hypothetical case that detection is not random with respect to other variables, e.g. epitope localization, and that non-randomness effects detectability. For an epitope assay system that yields a 30% coefficient of variation (normal or log-normal distributions), if the stimulated state is 5 to 10 times the unstimulated state, and the new method imparts a 66% reduction in the stimulated signal, the fractional increase and the MFI shift are completely resolvable by standard cytometric analyses of immunofluorescence as reported by Sladek and Jacobberger (21). We recognize that this may not be sufficient for some epitopes. In that case, the level of alcohol can be increased with a compromise on light scatter and level of expression of some surface markers.
At present, we do not have data that would indicate whether the fix-lyse (F/TX/MeOH) approach could provide a signal that is biologically equivalent to that of Method A (high MeOH). However, although we do not have evidence that our original technique provides biologically relevant measurements, we do know that the signal is equivalent to western blot analysis. In general, our experience indicates that fluorescence-based cytometric measurements are more precise but less sensitive than western blots (5, 16). Further, by comparing western blotting with cytometry for samples that have been extracted with various solutions (Frisa and Jacobberger, unpublished data), we have come to the expected conclusion that we detect only a fraction of the intracellular epitope by cytometry, i.e., a significant portion of the epitope is not available for staining after alcohol fixation/permeabilization and staining at antibody saturation. The effect of this situation is that the amplitude of response in an experiment is lower compared with western blotting, which is likely to be lower compared with the native state of the cell. Thus, we would argue that our improved method (F/TX/MeOH) is sufficient for the majority of studies in which we wish to preserve light scatter and surface immunophenotype (yet wish to rapidly stop a whole blood reaction), and in which our expected signal is sufficiently large above an appropriate background signal.
One benefit to alcohol treatment (as in the original method, or in F/TX/MeOH method from this study) is that it allows storage of fixed samples at −20°C for prolonged periods. Although our studies have only measured the impact of short-term storage (24 h with minimal change in pERK expression), a previously published study has suggested a 20% to 30% decrease in the measured levels of several different phospho-epitopes (as measured by flow cytometry) after as much as 5 months storage of samples in alcohol at −20°C (3). Frisa and Jacobberger (unpublished observations) measured decreases in each of several non–phospho epitopes in cells stored in 90% MeOH within a 6-month period. However, the loss of detectability is a complex process that appears to be a balance of chemical changes in the epitope under study and other masking elements; therefore, each epitope should be investigated individually before long-term storage.
In summary, we have reported the development of a protocol that allows processing of whole blood samples using an initial fixation step, followed by a detergent treatment step to permeabilize WBCs and lyse RBCs. A final methanol treatment step (which can be omitted, depending on specific assay requirements) improves the signal-to-noise ratio for pERK expression (and other phospho-epitopes, such as p-AKT and p-S6, manuscript in preparation) and can provide a medium for short-term sample storage at −20° C. The significant advantages of this protocol over previously reported techniques for the analysis of intracellular phospho-epitopes (1–8) include the initial fixation step, which maximizes the likelihood of phospho-epitope retention, the ability to use samples containing RBCs, and preservation of light scatter and representative cell surface (CD) determinants needed for clinical cytometry, while providing a detectable and acceptable pERK signal that is significantly above background levels.