Chapter 10

CLINICAL FLOW CYTOMETRY

A Transition in Utilization

Charles L. Goolsby, Mary Paniagua, Laura Marszalek

Department of Pathology (CLG), Robert H. Lurie Comprehensive Cancer Center (CLG and
MP), Northwestern University Feinberg School of Medicine, and Northwestern Memorial
Hospital (LM), Chicago, Illinois

1. INTRODUCTION

Over the last 25 years, flow cytometry analysis of hematopoietic

malignancies has evolved dramatically. In the early days, single color
analysis, or one antigen at a time, on ficoll-hypaque separated cells was the
standard approach. Thus, co-expression of antigens on cells, a hallmark of
current practice, could only be inferred by comparing numbers of cells
positive for each antigen. Density gradient preparation of the cells carried
with it the potential for either loss or enrichment of specific cell populations
that was variable from sample to sample. These technical limitations limited
the sensitivity and specificity of leukemia/lymphoma immunophenotyping in
those early days. Certainly, even with these limitations, however, these data
were still very useful as an adjunct to diagnosis, although at the time, seldom
were they integrated into the primary diagnostic package. Most frequently,
the immunophenotypic results were issued as a separate document,
frequently days after the primary diagnostic pathology report.
In the intervening years since those early beginnings, there has been
steady development of reagents and instrumentation. We have seen not only
dramatic increases in the numbers of highly specific monoclonal antibodies
but, particularly recently, in the numbers of fluorescent dyes available as
well. The standard of practice is now routine four color immunophenotyping
with many laboratories moving to five, six, seven or more colors. For the
most part, density gradient preparation of cells for these analyses is a thing
240 Clinical Flow Cytometry

of the past. These changes have dramatically increased the sensitivity and
specificity of these assays. The analyses have become the preferred method
of lineage determination in acute leukemias when morphology or special
stains are unable to make that assessment.1,2 Sub-classification of the B cell
chronic lymphoproliferative disorders now depends heavily on
immunophenotypic data,2 and cytometric analyses provide a sensitive
method of detection of clonal B cell populations.2 Routinely, laboratories
can detect malignant cell populations at significantly less than 1% of the
total cells (frequently less than 0.1%) and in some cases can approach RT-
PCR levels of sensitivity. In addition, it is now accepted that careful and
thorough correlation of these data with other pathology findings,
cytogenetics, and molecular analyses is critical for correct interpretation and
maximizing the appropriate utilization of these data.1,2 As a result, in most
institutions, immunphenotyping has become part of the primary diagnostic
work-up, ideally incorporated into the primary diagnostic reports. In
addition, to continuing to serve an increasingly important adjunctive role in
diagnosis, in some instances, immunophenotyping provides important
prognostic information as well, for example CD38 expression in HIV,3
CD38 expression in B-CLL,4-6 or Zap70 expression levels in B-CLL.7
Although not the topic of this chapter, the reader is referred to any of a
number of excellent reviews on the diagnostic and prognostic uses of these
data for more details.1,2 It is worth noting that the role as an adjunct to
diagnosis continues to evolve with new AML sub-classifications based on
signal pathway responses to growth factors being proposed.8 This is a very
attractive concept in that these measurements will most likely reflect at the
functional level abnormalities that are the result of disease characteristic
translocations and genetic abnormalities. Perhaps most exciting is that they
may then directly tie to new, and emerging, “targeted” therapies directed at
these abnormal signaling events that in many instances have lead to the
dysregulated proliferation and/or apoptosis that drive the disease process.

2. HISTORICAL PERSPECTIVE

In addition to its utility as an adjunct to diagnosis, exciting new horizons

for clinical cytometry are rapidly emerging, and developing, as tools in the
therapeutic management of leukemia and lymphoma patients. Increasingly
flow cytometric analyses are becoming key in therapeutic decisions and in
therapeutic monitoring of patients. The explosion of antigen and ligand
directed therapies for a range of hematopoietic malignancies, including anti-
CD20,9-12 anti-CD22,13 anti-CD52,11,12,14 anti-CD30,15 anti-CD33,11,12,16-18
anti-CD45,11,17,18 and IL-2,19 are having a significant impact on most clinical
Clinical Flow Cytometry 241

flow cytometry laboratories. Determining whether the malignant cells

express the relevant antigen or ligand receptor is key in determining patient
eligibility for a given therapy. Further, monitoring to see if the therapeutic
reagent has bound and whether that has resulted in cell death of both the
relevant malignant cells as well as normal cells is key in therapeutic
monitoring.9 The first major foray of flow cytometry into the arena of
therapeutic monitoring of an antibody treatment was in the setting of OKT3
immunosuppressive therapy in solid organ transplant patients.20-24 OKT3, an
anti-CD3 directed antibody, bound to the T cells leading to death of the T
cells and reductions or loss of cell mediated immune responses in these
transplant patients. Flow cytometric analyses employing a panel of
antibodies reacting with T cell antigens were used to determine if the drug
(OKT3 antibody) had bound to the T cells. Detection of T cells using
fluorescently labeled antibodies directed against either CD3 epitopes
independent of the OKT3 binding site, TCR, or other T cell associated
antigens such as CD2, CD5, or CD7 were employed along with a
fluorescently labeled OKT3 antibody. This allowed one to first, determine
on the T cells present (detected based on TCR, CD2, CD5, or CD7 staining)
whether the unlabeled OKT3 drug had bound since it would block binding of
the labeled OKT3 reagent being used. Further, it facilitated determination of
therapeutic efficacy by enumerating either, or both, the relative or absolute
numbers of T cells indicating whether the drug had successfully eliminated
the T cells. Flow cytometric bead based assays were also used to monitor
circulating levels of the OKT3 drug 21 and to determine if the patient was
producing antibodies directed against the treatment OKT3 antibody23,24
which might potentially be responsible for immunosuppressive treatment
failure. In addition, examination of T cells and T cell subsets was useful in
assessing T cell immune status following reduction or modulation of
immunosuppressive therapy.

3. CURRENT ROLE IN PATIENT THERAPEUTIC

MANAGEMENT

As noted above, the use of flow cytometric immunophenotyping as a tool

in therapeutic monitoring is growing, at least initially, primarily due to the
rapidly expanding numbers of antigen, or receptor, directed therapies that are
now options for treatment of a number of hematopoietic malignancies.9-19
The first impact of immunophenotyping is in determining whether a patient
is eligible for a specific targeted therapy through assessment of the relevant
therapeutic target (antigen(s) or receptor(s)) expression specifically on the
malignant cells. Many clinical flow cytometry laboratories now routinely
242 Clinical Flow Cytometiy

catalogue the expression of a number of potential therapeutically relevant

antigens on the malignant cells (CD20, CD22, CD25, CD52, CD30 for
example) for every relevant diagnostic specimen. In many instances, this
permits alternative treatment decisions to be made at a later time, potentially
without the need to re-biopsy the patient. In addition, as with OKT3
immunosuppressive therapy, flow cytometric immunphenotyping can be
useful in monitoring therapy by determining if the drug has bound and if that
has lead to reductions or loss of the malignant cells. Importantly, the effect
of drug on the patient’s normal cells expressing the same therapeutic target
(antigen or receptor) can also be assessed.
As an example, monitoring of rituximab (anti-CD20) therapy in patients
with a B cell malignancy is now routine and can play an important role in
monitoring of these patients.9 In a like manner to monitoring of OKT3
therapy described above, a panel of antibodies directed against B cell related
antigens including CD20, CD19, CD79b, kappa, and lambda, allows one to
determine if the anti-CD20 therapeutic has bound and whether the B cells,
more specifically the clonal B cells, have been eliminated in that patient.
With initiation of rituximab therapy, typically, a loss of peripheral B cells is
seen over 1-3 weeks.9,25,26 However, in some patients, one can see binding
of the drug but no loss of the clonal B cells.9 An example of this
demonstrating successful binding of the anti-CD20 therapeutic without loss
of the clonal B cells in a rituximab treated B cell lymphoma patient is shown
in Figure 1.
Flow cytometric analyses can also provide a sensitive tool for monitoring
of recurrence in these patients, a not always clear picture as morphologic
assessment of post-treatment bone marrows following rituximab therapy can
be complex. Immunophenotypic analyses can be a significant adjunct in
differentiating reactive responses from residual or recurrent disease in these
patients.9 In addition, it is worth noting that, although rare, recurrence of true
CD20 negative B cells of the same clone as the pre-treatment malignancy
have also been reported. Further, flow cytometric analyses provide a highly
sensitive detection of residual disease cells in the setting of antibody
mediated therapies.27 Although at present it is not always clear how to
interpret the finding of a small (<0.1%) clonal population, the potential for
detecting, and understanding, emergence of resistance in this setting
remains. This sensitivity may also prove useful in selecting and assessing the
effectiveness of additional, or co-, therapies. Again, although the potential
impact of small B cell clones may not be clear, screening of patients prior to
stem cell collection, or of collected stem cell products, during periods of
remission following antibody therapy is also frequently done. Lastly, again
in parallel with the OKT3 therapeutic monitoring model, flow cytometric
immunophenotyping can be useful in monitoring regeneration of the B cell
Clinical Flow Cytometry 243

compartment following cessation of therapy, as interestingly, return of

normal B cell levels in the peripheral blood can take many months. 9,25,26

Of note are reports of differential cell killing in different tissue

compartments following anti-CD52 therapy (Campath).28-30 In B cell non-
Hodgkin’s lymphoma, poor responses have been reported in lymph nodes as
compared to peripheral blood and bone marrow.29,30 Also, a case report of a
patient with a t(14;18)+ lymphoma in which the peripheral blood and bone
marrow remained PCR positive following chemotherapy showed eradication
of the B cell clone in the peripheral blood at the PCR level but not in the
bone marrow with Campath treatment.28 It is important to note in the latter
case that the bone marrow remained positive for lymphoma by morphology
and immunohistochemistry following Campath as well. This differential
response in different tissues to therapy has lead to clinical trials assessing the
244 Clinical Flow Cytometry

combination of rituximab and Campath in treatment of B cell

malignancies.31 Although significant clinical responses were seen, in initial
trials of rituximab addition to Campath treatment of B-CLL patients,
eradication of the clonal B cells was still not seen in the bone marrows of
these patients.32 Thus, in the immunophenotypic monitoring of patients
treated with Campath, and likely other antibody mediated treatments,
examination of not only peripheral blood, but in many patients, simultaneous
examination of bone marrow and tissue samples may be equally as
important.
Not unexpectedly, a similar testing scenario as that described above for
anti-CD20 therapy is being seen in the monitoring of other antigen directed
therapies. There is every reason to believe that the numbers of antigen and/or
ligand targeted therapies for the treatment of hematopoietic malignancies is
only going to grow over the next several years.10,33 In addition, an
increasing utilization of these therapeutic strategies is being seen in a variety
of other diseases and treatment settings.34-44 Thus, an increasing demand for
assessment of a large number of therapy related markers can be anticipated
in most clinical flow cytometry laboratories. These assays will probably
become more complex involving minimally four color analyses, and in many
cases more than four colors. This will allow more efficient analysis of the
large number of antigens and ligands that will be of interest to the clinician.
However, there are more important, and fundamental, reasons for an
extension to greater than three or four color techniques. In many instances,
assessment of two, three, or even four antigens is necessary to simply
specifically identify the aberrant cell in a sample. This may leave only one,
or in some cases no, fluorescence color to assess the presence or expression
of a potential therapeutic target on the malignant cells or to assess the effect
of the therapeutic on those same malignant cells. Thus, effective assessment
of therapeutic binding and effects is going to require an extension into more
complex analyses. Specifically assessing the effect of the drug on apoptotic
and proliferation regulatory pathways will lead to new understanding of the
mechanisms of drug action,45 and as importantly, perhaps mechanisms of
resistance. An additional complexity may arise from the need to quantify
antigen, or therapeutic target, levels. Golay et al,46 have shown that at least
in vitro the cytotoxic killing of B cells by rituximab correlates with CD20
staining intensity and, although not yet proven, it has been suggested that
this may be the case in vivo as well.47 Further, flow cytometric monitoring
may play an important role in assessing cytokine or other treatments
designed to modulate the expression level of a therapeutic target, potentially
increasing the effectiveness of a directed therapy.48,49 Thus, the need to
quantitatively measure target molecule levels is almost certainly going to be
important.
Clinical Flow Cytometry 245

4. FUTURE POTENTIAL ROLES

Perhaps even more exciting is the potential role that cytometric analyses
may play in assessing and monitoring of the new generation of therapies
such as imatinib mesylate (STI571, Gleevec)50,51 that more specifically target
abnormal or dysregulated proteins in the malignant or abnormal cells in a
patient. Driven by the Human Genome and Proteomics initiatives, the pace
at which genes and molecules associated with specific diseases are being
identified has greatly accelerated as has the understanding of these
abnormalities in the context of cell regulation and the biology of disease.
This knowledge will foster increasingly rapid development of new
therapeutics that, like imatinib mesylate, more specifically target
abnormalities or dysregulated functions in the malignant or abnormal cells.
Clearly, in treatment of human malignancies, it is believed that development
of rational therapies targeted at known abnormalities in tumor cells versus
normal cells will produce significant reductions in therapy-induced toxicity
compared to conventional chemotherapy. This hope was realized in the
treatment of chronic myelogenous leukemia patients with imatinib mesylate,
where extremely low toxicity was seen. With this in mind, especially for
cancer modalities, previously reliable laboratory endpoints often may not
provide adequate information about the effects of treatment during therapy.52
Not surprisingly, many of the new rationally designed, directed therapies
target molecules involved in signal transduction pathways that regulate cell
proliferation, apoptosis, differentiation, or angiogenesis.52-54 The
development of new, and novel, flow cytometry cell based assays to measure
the impact of therapy on these regulatory pathways, both in the malignant, or
abnormal, cells as well as in normal cells, will play a vital role in the
monitoring of these therapies as well as development of new therapies. Most
regulatory pathways controlling cellular proliferation and apoptosis involve
a series, or cascade, of alterations of the state (such as phosphorylation) of a
number of molecules, ultimately changing the expression of key proteins
such as cyclins, cyclin dependent kinases (CDKs), or apoptosis regulatory
proteins that regulate cell growth and cell death. Sophisticated multi-
parametric analyses assessing in specific cell sub-populations the targeted
protein(s) and the therapeutic as well as the levels and molecular states, such
as phosphorylation, of key intermediate molecules in these pathways that are
regulated by, and downstream of, the targeted proteins will be required.
Further, since multiple regulatory pathways control growth and death,
cellular response to drug and emergence of resistance may not be restricted
to a single pathway. Thus, the cellular proliferative and apoptotic responses
regulated by these disrupted pathways along with the key endpoint
246 Clinical Flow Cytometry

regulatory molecules such as cyclins or CDKs will also need to be monitored

in the same cell sub-populations as part of these analyses.
Imatinib mesylate therapy50,51 for chronic myelogenous leukemia (CML)
patients represents one of the first successful examples of a paradigm in
which a genetic abnormality associated with a disease is identified followed
by identification of the gene(s) involved, the characterization of the protein,
and development of a new drug specifically targeting this aberrant protein as
a therapy. As such it may serve as a model in many respects for the
development of not only new drug therapies but for the tools that will be
needed to monitor these types of therapies. CML is a chronic leukemia
characterized by the accumulation of abnormal myeloid cells harboring the
Philadelphia chromosome.55,56 The Philadelphia chromosome represents a
translocation between chromosomes 9 and 22 leading to a fusion between
the BCR and c-ABL genes. This leads to the production of a fusion protein,
BCR-ABL kinase, having abnormal kinase activity, which plays a key role
in the development and progression of the disease.55-57 Imatinib mesylate
represents a kinase inhibitor developed as a therapeutic for the treatment of
CML.50,51 It has high specificity for inhibition of the BCR-ABL kinase,
although at higher concentrations it does show inhibition of other kinases
such as c-kit and PDGF receptor. Initial response to imatinib mesylate has
been spectacular with most patients rapidly achieving a complete clinical
remission.50,51 However, a very high percentage of these patients relapse, or
develop resistance to imatinib therapy.50,51,58
Several roles for cytometric (cell based) monitoring of these patients, and
as a model other kinase inhibitor treated patients as well, may arise.
Interestingly, during escalation of dose in imatinib mesylate clinical trials,
only limited toxicity was seen. Thus, the paradigm of determining toxic drug
levels during clinical trials and to use that information in assessing relevant
treatment dose is less applicable with imatinib mesylate. As mentioned
above, this may be a common theme as more rational targeted therapies
become available. An alternative that may provide an adjunct to determining
effective doses in vivo will be monitoring the phosphorylation of key
molecules that are regulated through phosphorylation by kinases targeted by
the drug used,59 in the case of imatinib, the BCR-ABL kinase. The
development of antibody reagents highly specific for phosphorylated
epitopes on signal transduction protein intermediates will permit one, in
single cell measurements, to functionally assess whether a kinase upstream
of the protein intermediate being measured is active or not. Immediate
downstream phosphorylation targets of the BCR-ABL kinase which may be
suitable monitors of BCR-ABL kinase activity include Crkl as well as
members of the Jak-Stat pathway, 60-65 specifically Stat1 and Stat5-- targets
which BCR-ABL kinase is able to directly phosphorylate independent of Jak
Clinical Flow Cytometry 247

activation.60,64 Additionally, the autophosphorylation site on BCR-ABL

could also be included. An example of the cytometric application of these
reagents in a model system highlighting their specificity for detection of
phosphorylated Stat5 (p-Stat5) is shown in Figure 10-2. These data
supporting the specificity of these reagents comes from stimulation of
peripheral T cells66 with the cytokines, IL-2 and IL-4. IL-2 activates the Jak-
Stat pathway leading to phosphorylation of a number of Stat molecules
including Stat5,67 and T cells stimulated with IL-2 show significant
increased staining for p-Stat566 as opposed to unstimulated controls or cells
stimulated with the cytokine IL-4, which also stimulates the Jak-Stat
pathway but primarily other stat molecules and not Stat5.67,68 In the specific
case of monitoring BCR-ABL kinase activity, we, in collaboration with
several other laboratories (Clinical Cytometry Consortium; Drs. Hedley,
Jacobberger, and Shankey), have developed multi-parametric assays to
measure p-Stat5 in clinical specimens. As a model for this application, K562
cells, a CML cell line constitutively expressing BCR-ABL kinase and
having high level of expression of p-Stat5,60 were spiked into normal
peripheral blood mononuclear cells.
248 Clinical Flow Cytometry

Figure 10-3 illustrates the ability to detect the high levels of p-Stat5 in
the spiked K562 cells based on their expression of CD15 and high level p-
Stat5, and to modulate that expression in vitro with imatinib mesylate
treatment. In general, coupling the measurement of P-Stat5 with
immunophenotypic markers to identify or enrich for the abnormal malignant
cells allows rapid determination in vivo of whether imatinib treatment has
inhibited BCR-ABL kinase activity specifically in the malignant cells
through assessment of p-Stat5 levels. The potential for this approach looks
extremely promising and this assay is currently being implemented in
several imatinib mesylate clinical trial centers. Recent articles by Hedley et
al59 and Nolan et al69 demonstrates the applicability of these types of assays
in other signaling pathways as well. Specifically, in the work of Hedley et al,
monitoring of p-Mek and p-Erk, signaling intermediates in the MAP kinase
pathway, in the presence or absence of a raf kinase inhibitor, points to the
potential for the application of similar approaches in monitoring of patients
during clinical trials of this inhibitor as well.

The importance of single cell measurements, as opposed to bulk

measurements on cell lysates, in assessing phosphorylation, or activity, of
Clinical Flow Cytometry 249

key molecules in signaling cascades was also noted. It was clear that in this
experimental setting the MAPK pathway was either on or off,59 a
determination that would have been difficult using methods that assess
whole cell lysates to measure the same endpoints. Whether this on/off
signaling at the single cell level is also true in more physiological settings
will be of great interest and these techniques provide the tools to directly
address that question. Thus, single cell measurements of signaling
mechanisms critical to clinical monitoring of patients (as discussed in more
detail below) may also be crucial to understanding the more basic biology of
signaling processes as well.
Although potentially important to understanding the biology of signaling
processes and the effects of the kinase inhibitors used, how important is the
determination of these phosphorylation events on a single cell basis versus
more traditional assays employing cell lysates in a clinical setting? Some
argument can be made that during overt disease, when the malignant
population is the predominant population present, bulk methods employing
lysis of all cells and measurement of phosphorylated targets such as p-Stat5
in Western analyses or other techniques may be adequate. However, as
patients respond, and the malignant population decreases, utilization of bulk
assessment techniques will less reflect the therapeutic impact on the
malignant cells and more reflect the impact on the normal cells in the
patient. In fact, when morphologic and clinical remission is reached, bulk
methods will predominantly be a reflection of only the normal cell response.
Bulk methods will rarely be effective in identification of rare malignant cells
that are resistant to drug therapy or in the sensitive, early detection of
resistant cells before overt relapse occurs. These are two arenas in which
flow cytometry based single cell measurements will be exquisitely sensitive
in assessing phosphorylation, or activation state, of multiple proteins in rare
cells. These measurements could also play a key role in selection of the most
appropriate co-therapy when resistance to an initial kinase inhibitor drug
arises.
Further, single cell measurement techniques will almost certainly be
important in understanding the mechanisms of drug resistance in individual
patients. These techniques afford a sensitive assessment of re-activation of a
signaling pathway despite presence of drug. For example, in development of
resistance to imatinib mesylate therapy, in general, one sees continued, or
recurrent, high level expression of p-Stat5 in the presence of imatinib
mesylate58 due to either amplification of BCR-ABL or mutation of that locus.
During remission, flow cytometry techniques to measure rare, resistant cells
emerging early in development of resistance will almost certainly be a more
sensitive technique for monitoring development of imatinib resistance.
However, whether this will be useful in management decisions of CML
250 Clinical Flow Cytometry

patients where all patients develop resistance and combination therapies are
becoming commonplace remains to be seen. Nonetheless, this paradigm
applies more generally and undoubtedly will play a role in this arena at some
level in not only imatinib but other targeted therapies as well.
With appropriate reagents, single cell measurements may be a useful
adjunct in acquiring an understanding of the development of resistance to
targeted therapies. Was resistance due to inhibition of drug uptake, or
alternatively, to other mechanisms of drug inhibition? Development of
resistance may in some instances be more complex than simply altering drug
uptake or development of mechanisms to inhibit drug action with subsequent
re-activation of the relevant pathway. Regulation of cell proliferation and
apoptosis occurs through a number of alternative, and in some cases
redundant, pathways. Thus, in some instances, development of resistance
will arise through cells utilizing alternative signaling pathways to the one
which is blocked. Multi-parametric approaches coupling measurement of
specific inhibition of one pathway as described above along with
measurement of other relevant regulatory pathways and functional endpoint
measurements of cell proliferation, cell regulatory molecules (cyclins, cyclin
dependant kinase, etc), apoptosis, or apoptosis regulatory molecules may be
useful in identifying emergence of resistance even when the therapeutic drug
is still effectively blocking its target molecule. The ability to utilize these
techniques to simultaneously measure multiple pathways has clearly been
demonstrated.69 In addition, knowing what these responses are and
expression of which molecules have been altered may point to the
mechanism of resistance, to what other pathways may need to be monitored,
and to potentially appropriate additional therapies.
In addition, particularly when one moves outside of the realm of
hematopoietic malignancies, only rarely are single kinase abnormalities
going to be the primary, or only, abnormality responsible for disease.52 Even
in the setting of CML, these techniques may be key in assessing the effects
of combination therapies used with imatinib mesylate and helping to
determine on a patient specific basis what the most effective combination of
treatments may be. Although most CML patients in chronic phase respond to
imatinib, response in accelerated and blast crisis phase is more
heterogeneous. Before initiation of therapy, assessment of in vitro response
to imatinib may be useful in determining which of these patients are likely to
respond to imatinib in vivo.
Another exciting arena over the last several years has been development
of gene therapy options.70-73 Insertion and expression of specific genes into
abnormal cells designed to functionally correct the defect in these cells is an
exciting prospect. Initial forays into this arena have meet with some well
publicized problems, nonetheless, this approach remains promising and is
Clinical Flow Cytometry 251

rapidly evolving. The potential heterogeneity in expression of an inserted

foreign gene into the abnormal cells and its effectiveness in restoring normal
function or overcoming the effects of an abnormal protein lends itself to
cytometric techniques facilitating evaluation of transfected gene expression
and its effects on specific regulatory pathways and in specific cell sub-
populations that would be difficult by other technologies. Flow cytometric
approaches will play a role in the process of generation of transfected cells
for these therapeutic approaches. For some time, flow cytometry approaches
have been employed to measure the expression level of a transfected or
transduced gene into a cell.74-80 Sorting of cells can also be used in lieu of
drug selection to establish stable transfected cell lines as well as for selection
of cells having a specific expression level of a transfected gene (protein)
from the heterogeneous expression typically seen to generate cell lines
having homogeneous, typically high, expression levels.81
Far more exciting, however, is an elegant example of the use of flow
cytometry to monitor the effectiveness in correcting a regulatory defect by
induction of a normal transfected gene, in this case wild-type p53, into a
background with abnormal expression using an adenovirus delivery system
recently shown by Jaccobberger et al.82 Not only did these approaches prove
to be a sensitive way of confirming successful, high expression of the wild-
type p53, but more importantly, the sophisticated multi-parametric
approaches were able to clearly show that this protein was functionally
interacting with other cellular proteins to restore normal p53 function in
these cells. It is likely that cytometric approaches will play a significant role
not only in the laboratory aspects of generation and characterization of these
therapeutic agents but also in the monitoring of successful function and
maintenance of these cells in the patient.

5. SUMMATION

Over the last twenty years, cytometric evaluation of hematopoietic

malignancies has evolved from an adjunctive test which was secondary in
the diagnostic work-up, typically following by several days the primary
diagnostic work-up, to one which although still adjunctive to morphology is
now a key aspect of the primary diagnostic package. The maturing of the
technology in this setting is satisfying; however, more exciting are the
glimpses of the next evolution of this technology in a clinical setting,
perhaps first to be seen in the realm of hematopoietic malignancies. Rapid
movement of cytometric approaches into the arena of therapeutic
management and monitoring of patients is being seen. This movement
involves more complex multi-parametric approaches than previously have
252 Clinical Flow Cytometry

been employed in most clinical cytometry laboratories with five, six, seven,
or even more color analyses finding their way into the clinical setting. These
developments and needs are going to require cytometry technologists with
extensive experience and training along with laboratory directors,
pathologists, and clinicians who are knowledgeable about the analyses along
with interpretation and application of the results. Interpretation of these
experimental results will require sophisticated cytometry skills as well as a
detailed knowledge of the signaling and regulatory pathways in which the
measured molecules function. This is a very exciting time in clinical
cytometry, perhaps the most exciting time yet, but a challenge remains in
providing the appropriate training mechanisms and tools to ensure that these
analyses can be adequately performed in a wide range of clinical
laboratories.

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