|
99130217
Al-Saady
NM, Leatham EW, Gupta S, Kwan JTC, Eastwood JB, Seymour CA.
Monocyte expression of tissue factor and adhesion molecules: the link
with accelerated coronary artery disease in patients with chronic renal failure.
Heart 1999;81(2):134-40
OBJECTIVE:
To investigate the expression of monocyte tissue factor (MTF) and adhesion
molecules in patients with chronic renal failure (CRF) and to look for any
correlation with thrombin generation and Lp (a) lipoprotein. DESIGN: A study of
MTF expression and adhesion molecules, prothrombin fragments 1+2 (PTf1+2), an
index of thrombin generation, and lipoproteins in patients with CRF and in
normal control subjects.
BACKGROUND:
Patients with end stage renal failure have an increased risk of coronary artery
disease despite advances in therapy. Stimulated monocytes are potent activators
of blood coagulation through the generation of MTF, which was recently
implicated in the aetiology of acute coronary ischaemic syndromes. METHODS: MTF
expression and adhesion molecules were measured in whole blood using
immunofluorescence of monocytes labelled with anti-tissue factor antibody and
CD11b and c by flow cytometry. PTf1+2 and Lp(a) lipoprotein in plasma were
measured by enzyme linked immunosorbent assay (ELISA). PATIENTS: 70 patients
with CRF without documented coronary artery disease (30 patients with CRF
undialysed, 20 patients undergoing chronic ambulatory peritonea dialysis (CAPD),
and 20 undergoing haemodialysis (HD)), together with 20 normal controls, were
studied. RESULTS: The (mean (SD)) increased MTF of CRF (48.0 (29) v 33.3 (7.2)
mesf unit/100 monocytes in controls, p = 0.04) was more pronounced in patients
undergoing dialysis (HD 73.1 (32.8) (p < 0.003) and CAPD 62.8 (28.9) mesf
unit/100 monocytes, p < 0.04). MTF activity showed a positive correlation
with both PTf1+2 and serum creatinine (p < 0.003) but not with Lp(a)
lipoprotein. Lp(a) lipoprotein was significantly increased in both dialysis
groups compared with controls (p < 0.005) and non-dialysis CRF groups (p <
0.02). Monocyte adhesion molecule (CD11b) was significantly higher in all three
CRF groups than in the controls (p = 0.006).
CONCLUSIONS:
This study has demonstrated a hypercoagulable state in patients with CRF. This
was especially pronounced in the dialysis patients. These findings provide a
possible explanation for the increased cardiovascular and cerebrovascular
morbidity and mortality in these patients.
99273615
Schmidtke
G, Schmucker U, Brittinger G, Hoffkes HG. Comparative
flow cytometric study of clonal excess in leukaemic peripheral blood from
patients suffering from chronic lymphocytic leukaemia (B-CLL) by different
antibodies, staining techniques and the effects of blood storage.
Clin Lab Haematol 1999;21(2):103-12
This
investigation studied the effects of cell preparation methods,different antibody
panels and blood storage on antigen expression of abnormal B lymphocytes from
patients with B-CLL. Blood specimens collected in Heparin de novo were processed
by using conventional Hypaque-Ficoll density gradient centrifugation and whole
blood lysis. These were stored for
3 days at 4 degrees C, 24 degrees C and 30 degrees C. Although clonal excess was
detected by all antibody panels, significant differences could be observed in
terms of molecules of equivalent fluorochromes (MEF/MESF units). Evaluation of
‘weak and strong’ staining is dependent on the antibody panel used.
Immunofluorescent values for CD19 and CD45 were unchanged at 4 degrees C
and 24 degrees C but immunoglobulin staining showed best results when blood was
stored at 4 degrees C. Storage at 30 degrees C produced unreliable results.
Abnormal B lymphocytes should be analysed immediately after the specimen is
obtained. If shipment is necessary they should be kept at 4 degrees C. Surface
immunoglobulins are the 'antigens' most sensitive to storage alterations. Sample
alterations alone are sufficient to the correct classification of NHL,
especially in the case of low-grade NHL.
98444874
Chase
ES, Hoffman RA. Resolution of dimly
fluorescent particles: a practical measure of fluorescence sensitivity.
Cytometry 1998;33(2):267-79
Flow
cytometry is usually used to analyze subpopulations of cells, not simply to
measure the mean fluorescence level of a mixture. Thus, resolution or
coefficient of variation (CV) of dimly stained populations is the most
appropriate measure of fluorescence "sensitivity." Methods used to
measure sensitivity that are in routine use do not unambiguously and completely
determine the ability of a flow cytometer to resolve dimly fluorescent
populations from each other. Since fluorescence sensitivity depends on two
factors, background light (B) and detection efficiency (Q, the detected
photoelectrons per fluorochrome molecule on an analyzed particle), one cannot
uniquely define the operating condition of a flow cytometer with just one of
these factors. In general, it is not possible to define the ability of a flow
cytometer to resolve dim subpopulations by using a single number such as
"noise level" or "detection threshold"-the description
requires a "two-parameter" measure. A carefully characterized flow
cytometer was used to determine the inherent fluorescent CV of dimly fluorescing
beads. The fluorescence from the beads is also calibrated in terms of molecules
of equivalent soluble fluorophore (MESF). The beads with known inherent CV and
MESF provide a standard against which the instrument contribution to the CV of
dim fluorescence can be measured. By measuring the standard deviation (SD) of
the fluorescence histogram from unstained beads (noise) we obtain a second
measure of instrument performance. The bead CV and noise SD are a
sufficient pair of factors to determine the optical capability of a flow
cytometer to resolve dim subpopulations of particles. It is also possible to use
the measurements to calculate B and Q and use this information to predict the
shapes of fluorescence histogram distributions of dim particles.
98352580
Glasova
M, Konikova E, Stasakova J, Babusikova O. The
relationship of HLA-DR, CD38 and CD71
markers to activation, proliferation and differentiation of some human leukemia
and lymphoma cells. Neoplasma
1998;45(2):88-95
We
investigated the expression-percentage as well as MESF values ("molecules
of equivalent soluble fluorochrom" that represent approximately the density
of marker expression) of HLA-DR, CD71 and CD38 markers in some human leukemias
(ALL, AML, CLL, CML) and lymphomas. They are non-lineage restricted and are
supposed to be activation markers except for cases where they represent
pathological phenotype like HLA-DR in pre B-ALL, CD38 in some M0 AML or in
plasmocytoma or CD38 and CD71 in less mature T-ALL.
We used flow cytometry, immunofluorescent staining, DNA staining by
propidium iodide and quantification by calibration particles. We demonstrated
increased MESF values of HLA-DR compared with controls in all investigated
disorders, what could have a prognostic value. We demonstrated significantly
higher MESF values of HLA-DR in cALL (37,300-46,000) in comparison with AML
(9400-12,400), what could represent another important parameter when
distinguishing between these two groups of leukemia. In cells of CML patients
with lower CD38% and CD71% increased MESF values (5100 for CD38 and 7900 for
CD71), were found while in some T-ALL, AML and cALL patients with high
percentages of CD71 and CD38 there were lower MESF values what could indicate a
possible connection of higher stage of cell maturation with increased density of
CD38 and CD71 markers. We investigated possible relationship between percentage
of expression of HLA-DR, CD38 and CD71 and proliferation rate by DNA analysis of
the cell cycle. In a group of non-Hodgkin's lymphoma patients, there was no
significant increase of proliferation index of malignant cells compared with
control. The correlation between percentage of expression of mentioned
parameters and proliferation index was not significant. In one patient with
Burkitt's lymphoma we demonstrated significant increase of proliferation index
of CD71+ subpopulation compared with CD71- one, what indicates that in
aggressive form of NHL CD71 can be evaluated not only as activation but also as
proliferation marker.
98444861
Gratama JW, D'hautcourt JL, Mandy F, Rothe G, Barnett D, Janossy G, Papa S,
Schmitz G, Lenkei R. Flow
cytometric quantitation of immunofluorescence intensity: problems and
perspectives. European Working Group on Clinical Cell Analysis. [Review] [43
refs] Cytometry 1998;33(2):166-78
Quantitation
of immunofluorescence intensity serves to estimate the number of defined
molecules expressed on or in cells. Clinical applications of this diagnostic
tool are increasing, e.g., aberrant expression of various antigens (Ag) by
leukemic blasts or lymphoma cells, intensity of CD38 expression by CD8+
T-lymphocytes to monitor activation status, and intensity of CD62P to detect
platelet activation. In this report we discuss the quality-control measures
required for quantitation of fluorescence intensity, and we review seven
concepts that have been developed to quantify fluorescence intensity during the
past 15 years. Initial work
addressed the conversion of logarithmic channel numbers into units of relative
fluorescence. The design and use of calibration beads labeled with predefined
amounts of dye allowed instrument-independent expression of fluorescence
intensity in units of molecules of equivalent soluble fluorochrome (MESF). This
method was refined by the combined use of such standards with monoclonal
antibodies (mAb) conjugated 1:1 with phycoerythrin (PE), allowing translation of
fluorescence intensity into numbers of antibodies bound per cell.
Alternatively, the use of 1:1 PE-conjugated mAb under the assumption that
CD4+ lymphocytes reproducibly bind 50,000 CD4 mAb molecules was proposed to
convert units of relative fluorescence intensity into units of antibodies bound
per cell. The use of antibody-binding capacity as a surrogate marker for
quantification of Ag expression was addressed more directly by the development
of antibody-binding standards. The quantitative indirect immunofluorescence
assay is based on beads labeled with various amounts of CD5 mAb that calibrate
the binding of the secondary antibody in units of antibody-binding capacity.
Alternatively, goat anti-mouse-labeled calibration beads have been developed.
Published results obtained with the latter calibrators showed an
unexpected inaccuracy. The different ways in which calibrators and cells under
study bind mAb (i.e., Fab mediated versus Fc mediated) may have contributed to
this variation. Recently, the use of stabilized cell populations expressing Ag
in a specified range of concentrations has been proposed as an Ag-specific
calibration system of mAb binding. We identify several issues on the level of
instrumentation, reagents, and cells under study that should be solved to allow
standardization of quantitative assessments of immunofluorescence intensity.
[References: 43]
98444853
Henderson LO, Marti GE, Gaigalas A, Hannon WH,
Vogt RF Jr. Terminology and
nomenclature for standardization in quantitative fluorescence cytometry.
Cytometry 1998 Oct 1;33(2):97-105
Terminology
in any field is a complex mix of established conventions,
accepted usages, disputed terms, and occasional misnomers. The
terminology that has evolved for quantitative fluorescence cytometry
(QFCM) is especially multifarious, in part because QFCM encompasses a
range from subjective visual assessments to objective photon counts. Thus,
while descriptive terms such as "dim" and "bright" are still
quite
useful, quantitative terms such as "binding capacity" should be used
with collective understanding of their exact meanings. This article reviews
current usage and proposes definitions that, with refinement from suppliers
and users of QFCM technology, can provide the required clarity.
98444867
Powell MK, Whitfield W, Redelman D, Henderson LO, Vogt RF Jr.
Titration of a CD45-FITC conjugate to determine the linearity and dynamic
range of fluorescence intensity measurements on lymphocytes. Cytometry 1998 Oct 1;33(2):219-24
To
produce biologic calibrators for relative fluorescence intensity (RFI)
measurements, we stained leukocytes with serial dilutions of CD45-FITC
conjugate and processed them using our regular whole blood lysis procedure.
Cells were stained with conjugate concentrations ranging from twice
recommended to a million-fold lower. At the highest concentrations of
conjugate, the RFI reached a plateau near the top of the third decade, indicating
saturation of CD45 binding sites. As the concentration
decreased, the RFI declined in a highly linear relationship between the dilution
factor and the histogram channel number. For channel numbers corresponding
to the lowest percentiles of the RFI distribution, linearity persisted
down to the first half decade. The slope of this relationship revealed a
true dynamic range of 4.5 decades, which was comparable to the value
obtained with microbead standards calibrated in molecules of equivalent
soluble fluorochrome (MESF). Our results suggest that the lower limit of
linearity for fluorescence intensity from
fluorescein isothiocyanate (FITC)-stained lymphocytes is below 500 MESF
and that cellular autofluorescence is the major limiting factor in
detecting and quantifying FITC-specific staining. This procedure
provides an adroit way of characterizing the linearity and dynamic range
of measurements for quantitative fluorescence cytometry using exactly the same matrix, stains, and preparation methods as those used for cellular
analytes.
98444854
Schwartz A, Marti GE, Poon R, Gratama JW, Fernandez-Repollet E.
Standardizing flow cytometry: a classification system of fluorescence standards
used for flow cytometry. [Review] [35 refs]. Cytometry 1998 Oct
1;33(2):106-14
The
growing number of standards commercially available in the field of flow
cytometry makes it difficult to know which standards to use to obtain a desired
level of quality assurance. A classification system of fluorescence standards
has been
developed on the basis of their physical
characteristics. In turn, these physical characteristics determine the ability
of the specific standards to perform selected functions, such as alignment,
target referencing, compensation,
and calibration. Knowing the properties and limitations of specific standards
will help flow cytometer users to select the
appropriate
standard for the application that they will be performing,
especially
in regard to intra- and interlaboratory quality assurance. Common
protocols used in conjunction with specific classifications of reference
standards can provide unified analysis regions or window of analysis across
different instruments and/or laboratories. In addition, specific classifications
of calibration standards can help select those standards that will provide
independent and direct comparison of instrument performance parameters,
especially in studies involving multiple laboratories. Knowledge and
understanding of the classification system can guide flow cytometer users in
more efficient and accurate instrument setup and quality control when conducting
research, as well as clinical
applications. [References: 35]
98255199
Tsurusawa
M, Saeki K, Katano N, Fujimoto T. Bcl-2
expression and prognosis in childhood acute leukemia. Children's Cancer and
Leukemia Study Group [see comments]. Pediatr
Hematol Oncol 1998;15(2):143-55
Bcl-2
expression and its prognostic value were evaluated in 42 children with acute
leukemia. The Bcl-2 expression of the leukemic blast cells was measured
quantitatively by flow cytometry and was further analyzed by the simultaneous
immunostaining of Bcl-2 with the surface membrane antigens, DNA, Ki-67 antigen.
All of the cases showed a consistent expression of Bcl-2 protein; virtually all
leukemic lymphoblasts were Bcl-2 positive.
Although the expression of Bcl-2 varied widely from 7 to 80 x 10(3) MESF
units, no significant difference was found in the mean value between the
patients with acute lymphoblastic leukemia and those with acute myeloblastic
leukemia. In more than half of the patients with AML, intraclonal heterogeneity
of Bcl-2 expression was observed. The
expression of Bcl-2 showed no apparent fluctuations during the different phases
of the cell cycle. However, the proportion of Bcl-2-positive and -negative cells
during the cell cycle was different between ALL and AML patients. In the ALL
patients, few Bcl-2-negative cells were detected only in the GI phase, whereas
in the AML patients Bcl-2-negative cells were detected in the S and G2/M phases,
as well as in the G1 phase. No
apparent difference was found in Bcl-2 expression between the Ki-67-negative
noncycling population and the Ki-67-positive cycling population. Of the clinical
features of these patients, only CD34 expression in the ALL patients was
associated with high levels of Bcl-2 expression. In the 28 untreated cases of
ALL, high expression of Bcl-2 was not an unfavorable factor for the outcome of
this disease.
98444858
Zenger VE, Vogt R, Mandy F, Schwartz A, Marti GE.
Quantitative flow cytometry: inter-laboratory variation.
Cytometry 1998 Oct 1;33(2):138-45
Quantitative
flow cytometry (QFCM) offers a means of standardization within and
between flow cytometers. QFCM parameters were set by determining the
antibody-binding capacity (ABC) of CD4, CD8, and CD3 cells from 10 normal
donors with the use of eight FACScan flow cytometers. QC3 beads and a
certified blank bead were used to set up the instruments. Fluorescein
isothiocyanate (FITC) conjugated to molecular equivalents of soluble
fluorochrome (MESF) microbead standards was used before and after the
donor samples were run to ensure that the machines were operating
consistently. Lyophilized cells (Cytotrol) were used as a
target, to control for antigen expression in the cell preparation. Quantitative Simply Cellular
(QSC) beads were used to establish a
standard
calibration curve for each of the FITC and phycoerythrin antibody
conjugates on each of the instruments. Single-parameter fluorescent
histograms derived from list-mode files were used to calculate the slope
(coefficient of response), intercept (zero channel),
number of channels per decade, and ABC or MESF threshold (blank bead).
The fluorescence intensity (geometric mean) of the positive and
negative
donor cell populations was compared with the standard curves, and the ABCs
were calculated. The results show consistent instrument performance between
laboratories. However, after standardization of CD3, CD4, and CD8 ABCs to
microbeads, large variations were noted between donors and laboratories.
The source of this variation does not appear to be in the instrumentation
but may be due to the lack of an unified set-up protocol, introducing
issues of antibody saturation, methods for whole blood lysis and
fixation, and the behavior of the microbead standards.
99039557
Zhou
M, Gu L, Yeager AM, Findley HW. Sensitivity to Fas-mediated apoptosis in pediatric acute
lymphoblastic leukemia is associated with a mutant p53 phenotype and absence of
Bcl-2 expression. Leukemia
1998;12(11):1756-63
Fas
(APO-1/CD95) is a cell-surface protein that can mediate apoptosis upon specific
ligand or antibody binding. The Bcl-2 protein may function as a modulator of Fas-induced
apoptosis by blocking a downstream activation step, and Bcl-2 expression in
acute lymphoblastic leukemia (ALL) cells appears to depend partly on expression
of a wild-type (wt) p53 tumor suppressor gene (Findley et al, Blood 1997; 89:
2986). We therefore investigated the relationship between sensitivity to Fas-mediated
apoptosis and (1) Fas expression, (2) p53 status, and (3) Bcl-2 protein levels
in pediatric ALL cell lines and primary leukemic cells. Cell lines included 21 B
cell precursor (BCP)-ALL and four T-ALL lines; in five cases, cryopreserved
primary leukemic cells from which these lines were established were also
examined. Additionally, we evaluated the effect of anti-Fas monoclonal antibody
on the activation of protease CPP32 and induction of apoptosis in these lines.
By SSCP analysis and DNA sequencing, we detected p53 mutations (mt) in eight out
of 25 ALL cell lines (exon-7, codon 248 n=6; exon-8, codon 273, n=2).
The expression of Fas and Bcl-2 was examined by immunofluorescence
staining and quantified as the number of molecules of equivalent soluble
fluorochrome (MESF). Elevated levels of Fas were expressed in all six lines with
a mutation of p53 in codon 248 (1500 to 10800 MESF). Although Fas was detectable
in seven of the 17 lines with wt-p53, expression was lower (150-900 MESF)
compared with mt-p53+ lines. Bcl-2 was expressed in 10 of the 25 lines. Most
(9/10) wt-p53+ lines expressed Bcl-2, whereas only one of eight mt-p53+ lines
and no p53-null lines expressed this protein. Treatment of Fas-positive lines
with anti-Fas monoclonal antibody (200 ng/ml) for 6 h induced activation of
CPP32 and apoptosis in eight of 13 Fas+ lines. Sensitivity to Fas-mediated
apoptosis was associated with a mt-p53 phenotype and absence of Bcl-2
expression. Six of eight Fas+/Fas-sensitive
(S) lines were mt-53+/Bcl-2-, whereas only two Fas+/Fas-S lines were
wt-p53+/Bcl-2+; both of these latter lines expressed low levels of Bcl-2
compared to Fas-resistant lines. In contrast, four of five Fas+/Fas-resistant
(R) lines were wt-p53+/Bcl-2+; the exception was p53-null/Bcl-2- but expressed a
low level of Fas (150 MESF). Activation of the cysteine protease CPP32 and
cleavage of its substrate poly(ADP-ribose)polymerase (PARP) was also detected in
Fas-S but not Fas-R lines. We obtained similar results from both the primary
leukemic cells and the corresponding cell lines in five cases: overexpression of
Fas and Fas-sensitivity were present in mt-p53+/Bcl-2- but not wt-p53+/Bcl-2+
cells. These results suggest that some pediatric ALL cells expressing mt-p53+
may be sensitive to Fas-mediated apoptosis due to high levels of Fas expression
and lack of Bcl-2, and further suggest that molecular methods of activating Fas
may be useful for therapy of refractory ALL with the Fas+/mt-p53+ phenotype.
97287706
Berenbrink
M, Weaver YR, Cossins AR. Defining the volume dependence of multiple K flux pathways of
trout red blood cells. Am J Physiol
1997;272(4 Pt 1):C1099-111
The
volume sensitivity of different K flux pathways has been determined in trout red
blood cells subjected to volume perturbation. Gentle hyposmotic swelling induced
a K influx in a Cl-containing saline but not in NO3- or methanesulfonate (MeSF)-containing
salines, consistent with the activation of a Cl-dependent flux. Extreme
hyposmotic swelling led to larger K fluxes in all salines but with reduced anion
discrimination of the Cl-dependent flux. In contrast to these graded responses,
isosmotic swelling using ammonium chloride or beta-adrenergic
stimulation activated only Cl-dependent fluxes in an all-or-none fashion.
The relationship between the hyposmotically and isosmotically induced
pathways was studied by coactivation using either ammonium chloride or
isoproterenol with anisosmotic treatment. Cells in ammonium chloride-containing
hyposmotic salines showed no additive K flux over that induced by hyposmotic
treatment alone, indicating that the isosmotically induced Cl-dependent flux was
identical to the hyposmotically induced Cl-dependent flux. However, cells
coactivated by hyposmotic and beta-adrenergic treatment showed a small Cl-dependent
flux in addition to that induced by hyposmotic treatment alone. This small third
component was unaffected by anisosmotic treatment. We conclude that the major Cl-dependent
and Cl-independent K flux pathways are distinct and separate and that the former
has an anion dependence that varies with cell volume and a volume sensitivity
that varies with ionic strength.
97309418
Borowitz
MJ, Shuster J, Carroll AJ, Nash M, Look AT, Camitta B, Mahoney D, Lauer SJ,
Pullen DJ. Prognostic significance
of fluorescence intensity of surface marker expression in childhood B-precursor
acute lymphoblastic leukemia. A Pediatric Oncology Group Study.
Blood 1997;89(11):3960-6
This
report describes the prognostic significance of the intensity of surface
membrane antigen expression in a series of 1,231 children older than 1 year with
newly diagnosed B-precursor acute lymphoblastic leukemia
(ALL) treated on Pediatric Oncology Group (POG) treatment protocols.
All patients had dual-color flow cytometric immunophenotyping performed
at a central reference laboratory with a standard panel of monoclonal
antibodies. The flow cytometers used in the study were calibrated with a
standard fluorescence microparticle that permitted conversion of relative
fluorescence channels to standard units of mean equivalents of soluble
fluorochrome (MESF). In univariate analysis, fluorescence intensity of CD45 and
CD20 was significantly associated with event-free survival (EFS), whereas other
markers showed no significant correlation with outcome. Patients whose blasts
were greater than the 75th percentile of intensity for CD45 (corresponding to
18,000 MESF units with CD45-FITC, or about 8% of the intensity of normal
lymphocytes) fared significantly worse than those with lower-density CD45, and
those whose blasts were greater than the 25th percentile of intensity
for CD20 (corresponding to 17,900 MESF units with CD20-PE) had a poorer EFS. The
intensity of both CD45 and CD20 was independently correlated with outcome.
There was no significant correlation between intensity of expression of
either antigen and traditional clinical risk factors, ploidy, or t(9;22) or
t(1;19). All patients with t(4;11) had CD45 intensity greater than the 75th
percentile, but CD45 intensity retained its prognostic significance after
adjusting for t(4;11). In multivariate analysis, both CD45 intensity greater
than the 75th percentile and CD20 intensity greater than the 25th percentile
were significantly correlated with poor outcome independently of previously
reported poor prognostic factors including National Cancer Institute (NCI) risk
group, ploidy, trisomies of 4 and 10, and adverse translocations including
t(1;19), t(9;22), and t(4;11). We
conclude that in childhood B-precursor ALL, the intensity of expression of CD20
and CD45 provides prognostic information not available from simple consideration
of antigen expression as positive or negative, and adds to that obtained from
traditional clinical and biologic risk factors.
97206091
Bradbury
DA, Zhu YM, Russell NH. Bcl-2
expression in acute myeloblastic leukaemia: relationship with
autonomous growth and CD34 antigen expression. [Review] [48 refs]
Leuk Lymphoma 1997;24(3-4):221-8
The
bcl-2 gene encodes a mitochondrial protein that inhibits the onset of apoptosis
induced by growth factor withdrawal or cytotoxic agents.
Using quantitative flow cytometry and expressing bcl-2 levels as the
number of molecules of equivalent soluble fluorochrome (MESF) per cell, we have
shown that bcl-2 protein expression in the blast cells from patients with acute
myeloblastic leukaemia (AML) is heterogeneous, but not related to FAB type. The
blast cells from AML patients with the capacity to grow and survive autonomously
in vitro were found to have higher bcl-2 MESF values than those that were
dependent upon exogenous growth factors. We have previously reported that the
blast cells from 70% of AML patients exhibit autonomous growth and autocrine
growth factor production in vitro and that this has been shown to be an
important indicator of poor prognosis in AML. High bcl-2 expression has also
been associated with a low complete remission rate and poor survival in AML. In
the patients whose blast cells exhibited autonomous growth, neutralisation of
endogenous GM-CSF resulted in down-regulation of bcl-2 protein, whereas in blast
cells from patients whose cells proliferated only in the presence of added
growth factors, incorporation of recombinant human (rh) GM-CSF in the culture
media resulted in up-regulation of bcl-2. Because CD34 positivity has been
reported as another indicator of poor prognosis in AML, we compared bcl-2
expression in cases of CD34 positive AML, CD34 negative AML and CD34 positive
normal bone marrow cells. Bcl-2 was found to be strongly expressed on the CD34+
normal bone marrow cells. The blast cells from CD34+ AML patients expressed
significantly higher bcl-2 levels than CD34- AML patients.
In five cases of CD34+ AML, the bcl-2 levels were determined on purified
CD34+ and CD34- blast cell populations. The CD34+ blast cells were found to
express significantly higher bcl-2 levels compared with the CD34-blast cells.
Our data would suggest that quantification of bcl-2 in AML blast cell may be
useful as a prognostic indicator in AML. [References: 48]
97209437
Gratama
JW, Kraan J, Adriaansen H, Hooibrink B, Levering W, Reinders P, Van den Beemd
MW, Van
der Holt B, Bolhuis RL. Reduction
of interlaboratory variability in flow cytometric immunophenotyping by
standardization of instrument set-up and calibration, and standard list mode
data analysis. Cytometry
1997;30(1):10-22
Two
workshops addressed the question to which degree standardization of instrument
set-up and calibration, and standard list mode data analysis would reduce
interlaboratory variability of flow cytometric results on prestained peripheral
blood mononuclear cells (PBMC). Standard instrument set-up included uniform
positioning of the "windows of analysis" for the forward and sideward
light scatter and fluorescence (FL) 1 (i.e., fluorescein isothiocyanate [FITC])
and 2 (i.e., phycoerythrin [PE]) parameters. Reference standards and PBMC,
double-stained with FITC- and PE-conjugated monoclonal antibodies covering a
wide range of FL intensities and coexpression patterns, were sent out to 25
laboratories in Workshop 1 and to 35 laboratories in Workshop 2 with the
following requests: a) to set up instruments according to local and standard
protocols, b) to acquire list mode data on the PBMC with both instrument
settings, and c) to analyze both datasets according to local protocols.
Standard analysis of the list mode data acquired with uniform instrument
settings was performed centrally using so-called "latent class model"
software (Van Putten et al., Cytometry 14:86-96, 1993). This software provides
an automated, "no-gating" analytical method of lymphocyte
immunophenotypes and employs fixed FL marker settings as defined prior to each
analytical run. In Workshop 1, these markers were set in identical histogram
channels for all instruments based on results obtained with a reference
instrument. Standard analysis of list mode data acquired after uniform
instrument set-up led only to a 13% reduction of interlaboratory variability of
results as compared to data analysis using local protocols. The standard
protocol for instrument set-up led to uniform positioning of relatively strong
FL signals but variable positioning of unstained cells on the FL histogram
scales. Hence, standard FL marker settings were inappropriate for some
instruments. Therefore, instrument responses to FITC and PE signals in Workshop
2 were calibrated using microbeads labeled with FITC or PE in a range of
predefined FL intensities expressed in MESF units (molecules of equivalent
soluble fluorochrome). That approach allowed the positioning of the FL markers
for the standard analysis on the basis of identical FL1 and FL2 intensities,
expressed in MESF units, for all instruments. Standard analysis of list mode
data acquired after uniform instrument set-up and calibrated FL marker settings
led to a 43% reduction of interlaboratory variability as compared to data
analysis to local protocols. We conclude that standard list mode data analysis
using fixed FL marker settings reduces the interlaboratory variability of flow
cytometric results on prestained PBMC, provided that the instruments have been
set up in a uniform way and that FL markers have been standardized on the basis
of calibration of each instrument's response to the corresponding FL signals.
97464471
Lanza
F, Castagnari B, Rigolin G, Moretti S, Latorraca A, Ferrari L, Bardi A, Castoldi
G. Flow cytometry measurement of
GM-CSF receptors in acute leukemic blasts, and normal hemopoietic cells.
Leukemia 1997;11(10):1700-10
A
quantitative analysis of expression levels of GM-CSF receptors was performed by
flow cytometry in different disease categories, ie AML (n = 72), ALL (n = 18),
and MDS (n = 12), as well as 12 healthy volunteers, using three different
unconjugated GM-CSF/R monoclonal antibodies (McAbs) (HGM-CSFR (CD116), M5D12,
4B5F5), and appropriate standards. By using the reference HGM-CSFR McAb, in
healthy subjects we found detectable levels of GM-CSF/R on blood monocytes (mean
MESF (molecules of equivalent soluble
fluorochrome)/cell: 36.1 x 10[3]), neutrophils (mean MESF/cell: 7.4 x 10[3]),
bone marrow (BM) myelo-monocytic precursors (MESF range for the myeloid
component, ie promyelocytes, myelocytes, metamyelocytes: 11.7-40.5 x 10[3], and
for the monocytic lineage: 25.7-69.2 x 10[3]), and in two distinct subsets of BM
CD34+ progenitor cells (GM-CSF/R dim: 2.5 x10[3] MESF/cell, GM-CSF/R bright (10%
of the total number of CD34 cells: 22.0 x 10[3] MESF/cell). In these subjects,
there was no correlation between the expression levels of GM-CSF/R and CFU (CFU-GM,
CFU-GEMM, BFU-E) colony production. Among the AML
samples, M5D12 McAb was positive in 33%, 4B5F5 McAb in 90%, and HGM-CSF/R McAb
in 78% of the cases examined (range of MESF/cell for the HGM-CSFR McAb: 0.9 x
10[3]-106.7 x 10[3]). The highest MESF values were seen in the M5 FAB subvariety
(mean: 39.4 x 10[3]), where all the patients tested (n = 20) showed a strong
positivity for the HGM-CSFR McAb. On the contrary, all ALL samples were GM-CSF/R
negative except in two patients, who displayed a dim GM-CSF/R positivity (My+ALL:
1.3 x 10[3] MESF/cell; pro-B ALL: 1.0 x 10[3] MESF/cell). In most (>70%) M1
FAB subtypes, GM-CSF/R+ blasts co-expressed CD34low, HLA-DRhigh, CD33, CD38
antigens, and had little or no capacity to form CFU-GM colonies. GM-CSF/R+
blasts from the M5 FAB category were also positive for CD14, CD11c, CD33 and
CD87. Furthermore, the number of GM-CSF/R expressed by leukemic cells from five
out of 72 (7%) AML patients was above the highest values seen in normal samples
(>69.2 x 10[3] MESF/cell), allowing the possibility of using this marker for
the monitoring of the minimal residual disease (MRD) in a subset of AML. Cell
culture studies aimed at evaluating GM-CSF receptor modulation following AML
blast exposure to rhGM-CSF showed two distinct patterns of response; in the
first group (6/10 cases) rhGM-CSF down-modulated GM-CSF receptors, whereas in
the second group (4/10 cases), rhGM-CSF treatment was associated with either an
increase or no change in the number of GM-CSF/R. In conclusion, cellular GM-CSF/R
expression was variable and ranged from undetectable (ALL and a minority of AML)
to very high intensities in M5 AML, and were also documented in some M0 AML,
thus suggesting the concept that GM-CSF/R detection may be of help in lineage
assignment of undifferentiated forms. Since
the number of GM-CSF/R on AML blasts may be modulated after GM-CSF treatment, it
can be postulated that the clinical use of GM-CSF in this disease may be
optimized by a dynamic analysis of the number and the affinity status of GM-CSF-R
in blasts and normal hemopoietic cells.
97365229
Lanza
F, Latorraca A, Moretti S, Castagnari B, Ferrari L, Castoldi G.
Comparative analysis of different permeabilization methods for the flow
cytometry measurement of cytoplasmic myeloperoxidase and lysozyme in normal and
leukemic cells. Cytometry
1997;30(3):134-44
Using
a direct one-color (fluorescein isothiocyanate; FITC) staining method with a
Facscan flow cytometer, we evaluated the intracellular expression of two
granular constituents of myeloid cells [myeloperoxidase (MPO) and lysozyme] on
leukemic cells from 21 patients with acute myeloid leukemia (AML), and 6
patients with acute lymphoblastic leukemia (ALL).
Three different permeabilization techniques were used [FACS Lysing
Solution (FLy), B.Dis; Ortho-PermeaFix (OPF); Fix and Perm (F&P), Caltag]
prior to monoclonal antibody (McAb) staining, in order to verify the specificity
and the sensitivity of the three labelling methods towards the two model
antigens. Peripheral blood cells from 15 healthy subjects and Ortho Absolute
Control served as controls. Data were expressed as percentage of positivity, net
fluorescence intensity, ratio between mean fluorescence intensity (MFI) of
positive cells and that of isotypic controls (P/N ratio; evaluated in both
geometric and arithmetic scale), and, in 12 representatives cases (7 AML, 5
normal samples), in the form of both molecules of equivalent soluble
fluorochromes (MESF) and antibody binding capacities (ABC). As far as the
antigenic expression of MPO and lysozyme in normal samples is concerned, F&P
resulted, in our hands, in the most specific and sensitive staining, followed by
FLy solution and OPF, which showed positivity for MPO, and, to lesser extent,
for lysozyme in a considerable manner of lymphocytes (means 64% and 54%,
respectively, for OPF and FLy; range of ABC/cell: 0.9-5.2 x 10(3)) obtained from
healthy subjects. With the reference F&P permeabilizing solution, 90% and
80% of FAB M1-M5 cases were found to be positive for MPO and lysozyme,
respectively. However, M1, M2, and M3 AML FAB (French-American-British)
subvarieties were characterized by a brighter expression for MPO (mean ABC/cell:
89 x 10(3)) than that of lysozyme (mean ABC/cell: 12.5 x 10(3D)), whereas blast
cells from patients with M5a FAB subtypes showed higher levels of lysozyme (mean
ABC/cell: 65 x 10(3)) than that of MPO (mean ABC/cell: 0.1 x 10(3)). One of five
cases of FAB MO AML showed a dull positivity for MPO-7 McAb. Patients with ALL
were MPO and lysozyme negative using both F&P and FLy reagents, although a
certain degree of positivity was documented in some cases with OPF. Taking these
data together, it can be stated that the use of anti-MPO McAbs may be of great
value for the diagnosis and monitoring of acute leukemia and, along with
lysozyme McAb,
can provide useful information in the distinction of myeloid from monocytic
leukemias and in the lineage assignment of apparently biphenotypic forms.
However, the methodology used for the detection of these myeloid-associated
antigens is critical for a correct interpretation of cytofluorimetric data and
should be taken into account when evaluating data coming from multicenter trials
dealing with leukemias. A standardization of cytofluorimetric analysis of
intracellular antigens is needed in order to improve the reproducibility and
comparability of results in multicenter studies.
96424368
Bradbury
DA, Aldington S, Zhu YM, Russell NH. Down-regulation
of bcl-2 in AML blasts by all-trans retinoic acid and its relationship to CD34
antigen expression. Br J Haematol
1996;94(4):671-5
High
levels of expression of the bcl-2 oncoprotein in acute myeloblastic leukaemia
(AML) cells have been associated with low complete remission rates and poor
survival. The sensitivity of AML blasts to drugs such as Ara-C can be increased
by the down-regulation of bcl-2 expression by antisense oligonucleotides.
All-trans retinoic acid (ATRA) has been reported to increase the sensitivity of
AML cell lines to Ara-C and to induce differentiation in the HL60 promyelocytic
cell line, with both effects being accompanied by a decrease in bcl-2
expression. Using flow cytometry and a monoclonal antibody to bcl-2, we have
investigated the effects of ATRA (1 microM) on bcl-2 expression in the blast
cells of 25 AML patients and the K562 cell line after incubation for 72 or 24 h,
respectively. Using Kolmogorov-Smirnov statistical analysis where a D
value of > 0.12 was statistically significant, we found that in
8/25 AML samples and the K562 cells there was a significant decrease in bcl-2
protein expression after incubation with ATRA (D value range 0.14-0.44).
The mean peak fluorescence (MPF) values for the bcl-2 levels of the ATRA
responders (n = 8) was reduced to 35.5 +/- 6.9 following incubation with ATRA
compared to 47.6 +/- 8.2 (mean +/- SEM) for control samples incubated in the
absence of ATRA (P = 0.014). There was no significant difference between the
baseline bcl-2 molecules of equivalent soluble fluorochrome (MESF) levels in the
ATRA responders (48.9 +/- 5.7, n = 8) and the non-responders (41.3 +/- 3.9, n =
17) (mean +/- SEM) (P = 0.28). The down-regulation of bcl-2 expression by ATRA
was particularly associated with CD34-negative AML and of the eight AML
patients' cells that responded to ATRA by down-regulating bcl-2, seven were CD34
negative (P < 0.05). Our data suggest that the addition of ATRA to
combination chemotherapy would increase the chemosensitivity of some patients
with AML, particularly CD34-negative AML, due to down-regulation of bcl-2
expression.
96234340
Campos
L, Sabido O, Sebban C, Charrin C, Bertheas MF, Fiere D, Guyotat D.
Expression of BCL-2 proto-oncogene in adult acute lymphoblastic leukemia. Leukemia 1996;10(3):434-8
The
products of the BCL-2 gene prolong survival of lymphohematopoietic cells by
inhibition of programmed cell death. We studied bcl-2 protein expression in a
series of 43 adult acute lymphoblastic leukemia (ALL) at diagnosis, using a
specific monoclonal antibody and flow cytometry.
All samples expressed bcl-2 with a mean percentage of positive cells of
77.9. The level of bcl-2 in positive cells expressed as mean
equivalent of soluble fluorescence (MESF) was highly variable ranging from 5 x
10(3) to 552 x 10(3) (mean +/- s.d.: 96.5 +/- 109 x 10(3)). Neither the percentage of positive cells nor bcl-2 MESF
levels were correlated with initial characteristics including blood counts,
immunological phenotype, or cytogenetics. The survival of leukemic cells
maintained in cytokine-free liquid culture was not correlated with bcl-2
expression. However, cells from ALL with higher white blood cell (WBC) counts,
with t(9;22) translocation, or expressing myeloid surface antigens exhibited
significantly longer survival in this culture system. The outcome after
intensive chemotherapy did not differ according to bcl-2 expression. Factors
associated with poor outcome included WBC counts, presence of t(9;22)
translocation, presence of myeloid antigens and prolonged survival of cultured
cells. These results indicate that high levels of bcl-2 are not associated with
distinct clinical or biological characteristics in ALL.
97133633
Chen
JC, Davis BH, Bigelow NC, Ceckowski C, Robinson J, Sounart-Miscovich C, Steel
KA. Flow cytometric HLA-B27 typing
using CD3 gating and molecules of equivalent soluble fluorochrome (MESF)
quantitation. Cytometry
1996;26(4):286-92
The
determination of HLA-B27 (B27) status is important in the diagnosis of
ankylosing spondylitis, Reiter's disease, and other arthropathies.
Flow cytometric (FCM) typing of B27 is a relatively new method which
allows for rapid turnaround time and low cost. However, different leukocyte
populations may manifest significant variation in staining intensity with B27
antibodies. Therefore, gating utilizing only light scatter properties of cells
may lead to high readings in some B27-negative samples. Fluorescence gating on
CD3+ cells were postulated as a means to eliminate these anomalies. Furthermore,
quantitative FCM measurements, as afforded through use of molecules of
equivalent soluble fluorochrome (MESF) units, can minimize day-to-day and
instrument-to-instrument variabilities in fluorescence measurements.
We compared
CD3 gating to light scatter gating and MESF analysis on 123 specimens in a
4-month period and found: 1) CD3 gating gave the lowest nonspecific B27 antibody
binding among B27-negative subjects; 2) there was no significant difference in
MESF values between CD3 gating and light scatter gating of B27-positive samples;
3) there was a wide range of B27 antibody binding fluorescence intensities among
B27-positive subjects; 4) identification of patients with low B27 expression may
require the use of CD3 gating in order to avoid costly confirmation testing; 5)
fluorescent standard beads are stable for routine use in a clinical flow
cytometry laboratory.
96151998
Coustan-Smith
E, Kitanaka A, Pui CH, McNinch L, Evans WE, Raimondi SC, Behm FG, Arico M,
Campana D. Clinical relevance of
BCL-2 overexpression in childhood acute lymphoblastic leukemia.
Blood 1996;87(3):1140-6
Enforced
BCL-2 gene expression in leukemic cell lines suppresses apoptosis and confers
resistance to anticancer drugs, but the clinical significance of increased BCL-2
protein levels in acute lymphoblastic leukemia (ALL) is unknown. Among 52
children with newly diagnosed ALL, BCL-2 expression in leukemic lymphoblasts
ranged widely, from 4,464 to 59,753 molecules of equivalent soluble fluorochrome
per cell (MESF), as determined by flow cytometry. The mean (+/- SD) level of
MESF in 43 cases of B-lineage ALL (19,410 +/- 11,834) was higher than that
detected in CD10+ B-lymphoid progenitors from normal bone marrow (450 +/- 314; P
< .001), and CD19+ peripheral blood B lymphocytes (7,617 +/- 1,731; P = .02).
Levels of BCL-2 in T-ALL cases (17,909 +/- 18,691) were also generally higher
than those found in
normal CD1a+ thymocytes (1,762 +/- 670), or in peripheral blood T
lymphocytes (9,687 +/- 3,019). Although higher levels of BCL-2 corresponded to
higher leukemic cell recoveries after culture in serum-free medium, they did not
correlate with higher cell recoveries after culture on stromal layers, or with
in vitro resistance to vincristine, dexamethasone, 6-thioguanine, cytarabine,
teniposide, daunorubicin or methotrexate. BCL-2 protein levels did not correlate
with presenting clinical features. Unexpectedly, however, lower-than-median MESF
values were significantly associated with the presence of chromosomal
translocations (P = .010). Notably, all six cases with the Philadelphia
chromosome, a known high-risk feature, had low levels of BCL-2 expression (P =
.022). Higher levels of BCL-2 were not associated with poorer responses to
therapy among 33 uniformly treated patients, and were not observed in three
patients studied at relapse. In
conclusion, increased BCL-2 expression in childhood ALL appears to enhance the
ability of lymphoblasts to survive without essential trophic factors, and is
inversely related to the presence of chromosomal translocations. However, it
does not reflect increased disease aggressiveness or resistance to chemotherapy.
96413431
DiGiuseppe
JA, LeBeau P, Augenbraun J, Borowitz MJ. Multiparameter
flow-cytometric analysis of bcl-2 and Fas expression in normal and neoplastic
hematopoiesis. Am J Clin Pathol
1996;106(3):345-51
Apoptosis
(programmed cell death) is an important regulatory mechanism in hematopoiesis,
and is thought to be a principal mechanism of action of cytotoxic chemotherapy.
Proteins that modulate apoptosis include bcl-2,
which inhibits apoptosis, and Fas (CD95, also known as APO-1), which induces
apoptosis in susceptible cells bound by Fas ligand (FasL). To characterize
precisely the expression of these apoptosis-regulatory proteins in normal and
neoplastic hematopoiesis, the authors have performed multiparameter flow
cytometric (FCM) analysis in a series of normal and abnormal marrow specimens.
Among normal hematopoietic elements, bcl-2 expression was highest in myeloblasts
(29 [+/- 9] x 10(3) molecules of equivalent soluble fluorochrome [MESF]), and
lymphocytes (28[+/- 7] x 10(3) MESF). bcl-2 was essentially undetectable in
granulocytes and nucleated red blood cells, whereas monocytes and B-cell
precursors expressed intermediate levels of bcl-2 (11[+/- 4] x 10(3) and 7[+/-
1] x 10(3) MESF, respectively). Fas expression increased with granulocytic and
monocytic differentiation; myeloblasts expressed 8(+/- 2) x 10(3) MESF, whereas
granulocytes (15 [+/- 2] x 10(3) MESF) and monocytes (28[+/- 5] x 10(3) MESF)
displayed relatively greater intensity of staining for Fas. Among lymphoid
cells, Fas expression was heterogeneous. B
cells expressed lower intensity Fas staining than both CD4+ and CD8+ T cells.
Myeloblasts in 30 cases of myeloid leukemia and myelodysplasia studied
for bcl-2 and/or Fas expression manifested variable levels of these molecules
(range 9-105 x 10(3) MESF for bcl-2 and 3-33 x 10(3) MESF for Fas). In addition,
intraclonal heterogeneity of bcl-2 and Fas expression was seen in certain cases
of AML, which correlated with extent of differentiation. Among 28 cases of
B-precursor ALL studied for bcl-2 and/or Fas expression, bcl-2 ranged from 22 to
60 x 10(3) MESF (P < .001 versus normal marrow B-cell precursors), and Fas
varied between essentially undetectable levels and 6 x 10(3) MESF. In summary,
normal marrow subsets display characteristic levels of the apoptosis-regulatory
molecules, bcl-2 and Fas. In hematopoietic neoplasms, expression of bcl-2 and
Fas varies among cases, and in some instances, within leukemic blast
populations. Further study is required to understand the potential significance
of this heterogeneous expression of bcl-2 and Fas in hematologic neoplasia.
97171581
Fletcher
A, Bryant JA, Stavliotis M. Coordinator's report on the flow cytometric analysis of the
Rh antibodies. Transfus Clin Biol
1996;3(6):407-13
One
hundred and four IgG monoclonal antibodies with specificity within the Rh Blood
Group System were evaluated by flow cytometry as part of the Third International
Workshop. Standardisation of data to permit interlaboratory comparison of
antibody binding was achieved by adherence to a standard red blood cell staining
protocol, defined control cells and a standard FITC-labelled antibody. In
addition, calibration standards were provided to permit the calculation of
Molecules of Equivalent Soluble Fluorochrome (MESF) values from the mean channel
fluorescence. For the majority of anti-D antibodies mean MESF values obtained
with D positive cells were far higher than with the negative controls (D
negative cells), with D variants having intermediate although very varied MESFs.
In general MESF values obtained with non anti-D antibodies were less than
for the anti-D antibodies although some of the anti-E antibodies were very
strong. The highest MESF values were obtained with an anti-CD antibody.
97056132
Frankel DS, Frankel SL, Binder BJ, Vogt RF.
Application of neural networks to flow cytometry data analysis and real-time
cell classification. Cytometry 1996 Apr 1;23(4):290-302.
Conventional analysis of flow cytometric
data requires that population identification be performed graphically after
a sample has been run using two-parameter scatter plots. As more parameters
are measured, the number of possible two-parameter plots increases geometrically,
making data analysis increasingly cumbersome. Artificial Neural Systems
(ANS), also known as neural networks, are a powerful
and convenient method for overcoming this data bottleneck. ANS "learn"
to make classifications using all of the measured parameters simultaneously.
Mathematical models and programming expertise are
not required. ANS are inherently parallel so that high processing speed
can be achieved. Because ANS are nonlinear, curved class boundaries and
other nonlinearities can emerge naturally. Here, we
present biomedical and oceanographic data to demonstrate the useful properties
of neural networks for processing and analyzing flow cytometry data. We
show that ANS are equally useful for human leukocytes and marine plankton
data. They can easily accommodate nonlinear variations in data, detect
subtle changes in measurements, interpolate and classify cells they were
not trained on, and analyze multiparameter cell data in real time.
Real-time classification of a mixture of six cyanobacteria strains was
achieved with an average accuracy of 98%.
98107486
Hoffkes
HG, Schmidtke G, Schmucker U, Brittinger G.
Lymphocyte gating of peripheral blood in patients with leukemic low-grade
non-Hodgkin's lymphoma by multiparametric flow cytometry.
Eur J Med Res 1996;1(4):215-22
The
standardized fluorescence intensity as expressed in molecules of equivalent
soluble fluorescence (MESF) of lymphocytes from normal individuals and patients
suffering from low-grade non-Hodgkin's-lymphomas was obtained comparing
different staining patterns of CD45(FITC) and CD20(PerCP). After standardization
of the flow cytometer using standardized fluorescent particles ('beads')
significant differences could be obtained for hairy cell leukemia, chronic
lymphocytic leukemia and immunocytomas in the peripheral blood. In contrast,
centroblastic-centrocytic as well as centrocytic lymphomas showed no significant
variations as compared to normal peripheral blood lymphocytes. According to
these results, a new lymphocyte gating procedure was established by adding
CD14(PE) and three-color measurement by CD45/CD14/CD20 staining of peripheral
blood using erythrocyte lysis. The established gating procedure leads to a crucial
discrimination and quantification of abnormal and normal lymphocytes per one
measurement, whereas the 'leucogate' as defined by CD45/CD14 staining alone was
insufficient for correct lymphocyte gate setting. In conclusion, the different
staining of CD45 and CD20 in leukemic peripheral blood should be considered when
fluorescence intensity or atypical peaks occurred in flow cytometric histograms
suggesting for abnormal cell populations. In addition, it is possible to use this information to
classify low-grade lymphomas.
97109926
Ocqueteau
M, San Miguel JF, Gonzalez M, Almeida J, Orfao A.
Do myelomatous plasma cells really express surface immunoglobulins?.
Haematologica 1996;81(5):460-3
Surface
immunoglobulins (sIg) are traditionally considered to be absent in plasma cells
(PC). However, it has recently been reported that up to one third of myeloma
patients are positive for surface immunoglobulins.
Nevertheless, this observation has not been confirmed in other studies.
In order to assess whether or not sIg are really expressed by myelomatous
PC and to exclude possible staining of either cytophilic or cytoplasmic Igs,
simultaneous experiments were carried out with i) incubation at 37 degrees C,
ii) blocking with non-conjugated anti-Ig light chains, and iii) cytoplasmic
staining after cell membrane fixation and permeabilization. Triple staining for
CD38/kappa/lambda was used in all cases and the staining intensity was
quantitated in MESF (molecule equivalents of soluble fluorochrome). In addition,
20 B-CLL cases and 10 healthy donors were used as reference controls. Our study
shows that 7 out the 20 patients (35%) analyzed expressed sIg and that the
surface staining was specific.
96209426
Ritzi
EM. Quantitative flow cytometry
reveals a hierarchy of glucocorticoid effect on cell surface mouse mammary tumor
virus gp52. J Steroid Biochem Mol
Biol 1996;57(1-2):33-42
A
flow cytometry protocol with CM mouse mammary tumor cells (Mm5mt/C1) was
utilized to provide a fluorescence measurement of hormone-mediated changes in
mouse mammary tumor virus (MMTV) cell surface envelope glycoprotein (gp52 CSA).
Standards permitted gp52-specific fluorescence intensity to be measured as
molecules of equivalent soluble fluorescein (MESF). The feasibility of using
MESF determinations
to reflect hormone-modulated changes in continuously infected cells was tested.
A panel of five glucocorticoids having differing affinity for the glucocorticoid
receptor were tested in 60 h treatments at dosages ranging from 10(-6) M to
10(-8) M. Determinations of MESF,
as a measure of gp52 CSA, were highest with 10(-6) M treatments (36.7-44.5 x
10(-6) MESF). At lower dosages, MESF determinations were lower but showed a
clear hierarchy of glucocorticoid effect. At 10(-8) M treatments, determinations
of MESF x 10(-6) demonstrated the following glucocorticoid hierarchy:
triamcinolone acetonide (TA) (33.7 +/- 1.6) > dexamethasone (DEX) (26.1 +/-
1.7) > prednisolone (8.0 +/- 0.3) > triamcinolone (6.6 +/- 0.4) >
hydrocortisone (6.4 +/- 0.4) > control (2.4 +/- 0.1). The MESF-derived
respective fold increases over control for this hierarchy were: 13.87, 10.74,
3.31, 2.71, and 2.65. The ability of TA to enhance gp52 CSA was 1.3-fold greater
than DEX. 10-fold higher levels of steroid controls did not significantly
elevate MESF levels. Findings argue that dosage, duration of treatment and
relative affinity of glucocorticoids for receptor are reflected in MESF
determinations of changing gp52 levels. Therefore, this new measure of effect
may be useful in studying hormonal influence on viral and cellular regulatory
systems in chronically infected cells.
96405337
Schwartz A, Fernandez Repollet E, Vogt R, Gratama JW.
Standardizing flow cytometry: construction of a standardized fluorescence
calibration plot using matching spectral calibrators.
Cytometry 1996 Mar 15;26(1):22-31.
Calibration
of flow cytometers is becoming an increasingly important issue for both
quality control of instrument performance and quantitation of antibody
binding capacity of cells. Due to the numerous different instruments and
analysis software currently available, a standardized method of
calibration is necessary if interlaboratory comparison of instrument
performance and antibody binding is to be achieved. This report describes
a new methodology to obtain a standard calibration plot that can be
derived from all instruments and from which specific instrument-independent
performance parameters may be calculated that can be used to directly
compare the performance and setup of these instruments. The requirements
that the calibrated standards must meet are discussed, as well as the
acceptable ranges proposed for the
instrument-independent performance parameters. In addition, data are
presented from standard calibration plots generated by different flow
cytometers in numerous laboratories. The corresponding Primary Performance Parameters calculated from these plots are presented and
compared.
It is expected that the use of this calibration method may help standardize
flow cytometric measurements and will provide instrument-independent
performance parameters to monitor quality control of instruments and
reagents.
96027661
Bradbury
DA, Russell NH. Comparative
quantitative expression of bcl-2 by normal and leukaemic
myeloid cells. Br J Haematol
1995;91(2):374-9
Expression
of the bcl-2 oncoprotein by AML blasts has previously been demonstrated to be
heterogenous with high levels of bcl-2 expression being associated with a low
complete remission rate and poor survival. We
have quantified bcl-2 expression in AML blasts in relation to expression of the
CD34 antigen and in comparison to CD34-positive cells from normal bone marrow.
When expressed as molecules of equivalent soluble fluorochrome (MESF) per cell.
AML blast cell bcl-2 expression varied from 11.1 to 99.9 x 10(3) (median 39.4 x
10(3), n = 56) with 28.5% of patients expressing high MESF values (> 50 x
10(3)) and 16% of patients expressing low MESF values (< 20 x 10(3), the
remainder expressing intermediate values. There was no significant difference
between intensity of bcl-2 expression and FAB classification in the de novo AML
cases; and there was no significant differences between de novo and secondary
AML cases. Blasts from CD34+ AML patients expressed significantly higher levels
of bcl-2 (mean MESF 43.6 x 10(3), n = 36) than CD34- AML patients (mean MESF
31.7 x 10(3), n = 19). In five cases of CD34+ AML, bcl-2 expression was
determined on purified CD34+ and CD34- blast cell populations. In all cases
CD34+ blasts were found to express significantly higher bcl-2 MESF values
compared to the CD34- fraction. Purified CD34+ cells from normal bone marrow
consistently expressed high levels of bcl-2 (MESF > 75 x 10(3), n = 4), which
was comparable to that found on CD34+ AML cells. Our results suggest that the
poor prognosis previously associated with AML blasts expressing the CD34 antigen
may in part be related to high expression of
bcl-2. Also the ability to measure bcl-2 in AML blasts quantitatively by
flow cytometry and to categorize patients into discrete groups may be of value
as a prognostic indicator in AML.
96106607
Chance
JT, Larsen SA, Pope V, Measel JW, Cox DL. Instrument-dependent
fluorochrome sensitivity in flow cytometric analyses [see comments].
Cytometry 1995;22(3):232-42
Flow
cytometry has become the preferred technique by which critical clinical
evaluations are made such as CD4 counts and aneuploid analyses.
Mounting concern has arisen over the numerous techniques, reagents, and
different flow cytometric employed to determine these data. Several studies have
documented significant differences in results when different flow cytometers are
utilized to analyze the same sample. Fluorochrome-dependent instrument
sensitivity also has been reported by numerous investigators. As more and more
procedures are performed by cytometric analysis, light scatter and fluorescence
limitations, which appear to be instrument dependent, demonstrate that not all
flow cytometers have the same capabilities. Attempts were made to calculate
molecules of equivalent soluble fluorochrome (MESF) values on nine different
flow cytometers using fluorescein isothiocyanate (FITC) and R-phycoerythrin
(R-PE) labeled microsphere reference standards produced by Flow Cytometry
Standards Corporation (FCSC). Dramatic differences were observed in the ability
of some cytometers to resolve these microspheres. The diminished resolution
appeared to be instrument model and fluorochrome dependent. We propose that
diminished fluorescence resolution in certain flow cytometers could be
responsible for significant variability in clinical values reported from
laboratories utilizing different flow cytometers.
96120903
Hudson
JC, Porcelli RT, Russell TR. Flow cytometric immunofluorescence and DNA analysis: using a
1.5 mW helium-neon laser (544 nm). Cytometry
1995;21(2):211-7
We
evaluate a 1.5 mW HeNe laser (544 nm) for use on an EPICS Elite with a 76
microns Sortsense flow cell. The two applications chosen were immunofluorescence
and DNA analysis. We measured the fluorescence threshhold of phycoerytherin
calibration beads to be approximately 336 MESF. Cell analysis with a HeNe laser
and Argon laser correlated well for the CD4PE, CD56PE, CD19PE conjugates, with
correlation coefficients of 0.98, 0.99, 0.94, respectively. The % positive and
mean channel fluorescence were comparable to the results obtained with a 15 mW
Argon laser. In addition, a three-color configuration yielded excellent results.
Cell analysis of CD4PE, CD3ECD and CD19Cy-Chrome with the HeNe laser and Argon
laser correlated well with correlation coefficients of 0.96, 0.95, and 0.92,
respectively. The histograms
showed good separation between the negative cells, the dimly staining cells and
the brightly staining cells. Propidium Iodide was chosen for DNA analysis. Full
CV values for whole blood DNA fluorescence using the green laser were good at
2.6%. These data indicate the low power 544 nm laser is sufficient to do
immunophenotyping and DNA analysis. Results may be explained by higher quantum
efficiency and lower background fluorescence. The wavelength of the 544 nm laser
is much closer to the excitation peaks of PI, PE, and the tandem dyes ECD and
Cy-Chrome. Also, the Raman scattering of water for the 544 nm laser has a longer
wavelength maximum than the emission peaks of PI, PE, and ECD. The major
advantages of this laser for the research laboratory are small size, no cooling
fan, low power requirements and low cost.
96077752
Lanza
F, Moretti S, Castagnari B, Latorraca A, Rigolin GM, Bardi A, Castoldi G.
CD34+ leukemic cells
assessed by different CD34 monoclonal antibodies.
Leuk Lymphoma 1995;18 Suppl 1:25-30
CD34
monoclonal antibodies (McAbs) are widely used to identify and isolate
hemopoietic progenitors and to classify acute and chronic leukemias. We assessed
the reactivity of 17 CD34 McAbs from the 5th International Workshop
on Leukocyte Differentiation Antigens with a variety of cells types: normal bone
marrow hemopoietic progenitors, 10 AML, 6 ALL, 11 CML. The reactivity for these
McAbs was compared with that of reference CD34 McAbs (Q-Bend 10 and 8G12). For
each cell population the % of McAb binding cells, the peak channel and the mean
fluorescence intensity (MFI) of the positive cells was evaluated. The peak
channel, the MFI and the number of positive cells varied significantly from case
to case, depending on the McAb and the type of leukemia. According to the
spectrum of reactivity three groups of McAbs were defined; however, 7 McAbs do
not belong to any of these subgroups. These groups were not entirely in
accordance with McAb classification based on enzyme cleavage that identified
three epitopes of the CD34 molecule. Some reagents were found to be more
specific for AML, other for ALL, CML or normal CD34+ cells. Normal bone marrow
light density cells showed a significantly higher percentage of positive cells
for 43A1 and MD34.2 McAbs compared to that documented for the remaining McAbs.
AML cells showed the most variable pattern of expression for the CD34 McAbs. In
leukemic samples, MESF (molecular equivalents of soluble fluorochrome) values
ranged from 18,200 to 322,000 and the number of binding sites per cells was
5,000-81,000.(ABSTRACT TRUNCATED AT 250 WORDS)
96116912
Mainou-Fowler
T, Craig VA, Copplestone AJ, Hamon MD, Prentice AG.
Effect of anti-APO1 on spontaneous apoptosis of B cells in chronic
lymphocytic leukaemia: the role of bcl-2 and interleukin 4.
Leuk Lymphoma 1995;19(3-4):301-8
The
cell surface protein apolipoprotein 1 (APO1) is expressed on various cell types
including malignant lymphoid cells. Triggering of APO1 protein with antibody (Ab)
induces apoptosis in APO1-expressing cells. We examined the effect of anti
(alpha) APO1 Ab on spontaneous apoptosis (SA) and bcl-2 expression in B cell
chronic lymphocytic leukaemia (B-CLL) in vitro. We also investigated the
anti-apoptotic activity of interleukin 4 (IL4) on the aAPO1-induced apoptosis in
B-CLL cells. Although expression of APO1 on B-CLL cells was not detectable by
immunofluorescence, alpha APO1 Ab induced apoptosis in these cells. At 24 hours
in culture the number of apoptotic cells was increased by a mean percentage (%)
of 27% (range: 21-38) in only half of the cases studied. But in all twelve cases
studied, at 48 hours alpha APO1 increased SA by a mean of 72% (range: 26-114) (P
< .001) and at 72 hours, the mean % increase was 69% (range: 31-96) in 6/7
cases (P < .001). This effect was alpha APO1 concentration dependent.
Interleukin 4 significantly protected B-CLL cells against alpha APO1-induced
apoptosis by a mean of 53% (range: 28-76) (P < .001). This protection was
specific to IL4 and it was significantly reduced or abolished with alpha IL4 Ab.
Expression of bcl-2 protein in untreated cultures was not significantly
different from that of the alpha APO1-treated cells; the mean equivalent of
soluble fluorochrome (MESF) (range) was 4.9 (3.0-6.8) and 5.2 (3.5-6.0)
respectively (P > 0.2). In fresh B-CLL cells the MESF (range) was 4.5
(2.4-6.6). Thus alpha APO1 Ab induced apoptosis in B-CLL cells by a pathway that
is independent of bcl-2 expression and partially blocked by IL4.
95165791
Mullen
PG, Windsor AC, Walsh CJ, Fowler AA 3rd, Sugerman HJ.
Tumor necrosis factor-alpha and interleukin-6 selectively regulate
neutrophil function in vitro. J
Surg Res 1995;58(2):124-30
The
neutrophil is an important effector cell of the host response to sepsis. Tumor
necrosis factor-alpha (TNF-alpha), a cytokine mediator of the septic response,
is rapidly released following endotoxemia or gram-negative bacteremia.
Interleukin-6 (IL-6) is another cytokine mediator of the host response to sepsis
whose role is less well understood than that of TNF-alpha. It is known to be
elevated in gram-negative sepsis, where peak levels have been correlated with
mortality. This study examined the effect of IL-6 alone and in combination with
TNF-alpha on three neutrophil functions--CD18 adhesion receptor expression,
phagocytosis, and superoxide anion generation.
Neutrophils from human volunteers were incubated with amounts of IL-6
ranging from 10 to 1000 ng/ml. At a concentration of 1000 ng/ml, IL-6 increased
neutrophil phagocytosis of opsonized bacteria (826 +/- 255 x 10(3) MESF vs 552
+/- 103 MESF, P < 0.05) and also increased neutrophil superoxide anion
generation (18.41 +/- 1.86 vs 12.6 nmol O2-/10(6) PMN/10 min, P < 0.05).
Lesser amounts of IL-6 had no effect on phagocytosis or superoxide generation.
IL-6 did not increase neutrophil CD18
adhesion receptor expression. Combining IL-6 with TNF-alpha at doses of 100 ng/ml
and 100 U/ml, respectively, neutrophil phagocytosis (221 +/- 455 MESF vs 552 +/-
103 MESF) and superoxide generation (23.18 +/- 1.86 vs 12.6 nmol O2-/10(6) PMN/10
min) were significantly (P < 0.05) increased above control by an amount
similar to that seen with 1000 U/ml TNF-alpha alone. (ABSTRACT TRUNCATED AT 250
WORDS)
96101993
Rigolin
GM, Lanza F, Castoldi G. Photomultiplier
voltage setting: possible important source of variability
in molecular equivalents of soluble fluorochrome (MESF) calculation?.
Cytometry 1995;20(4):362-8
We
evaluated the effect of flow cytometer photomultiplier (PMT) voltage setting on
molecular equivalents of soluble fluorochrome (MESF) calculation by using two
different quantitative microbead calibrating kits as calibrating standards and a
third one as an unknown testing sample of stable intensity of fluorescence.
Based on the analysis of residual values derived from linear regression and on
the determinations of the resolution index, we demonstrated that, on a FACScan
instrument, the window of photomultiplier setting for fluorescein isothiocyanate
(FITC) fluorescence determinations is 590-670 V.
However, the best region was in the range of 610-650 V. Within the
590-670 PMT voltage region, the evaluation of the same testing sample at
different PMT voltages, at 590 V, gave MESF percentage differences of 15% and
13% on the two lower unknown standards of intensity compared to the values
obtained at the nearby 630 V setting. In the analyzed PMT region, logarithmic
amplifier response was globally maintained, whereas evaluation of the same
unknown testing sample in both linear (Lin) and logarithmic (Log) amplification
demonstrated (at the higher intensities of fluorescence) Log-Lin minimal
differences within the 610-650 PMT voltage range. According to our data, it can
be stated that PMT voltages between 610 and 650 provide the best instrument
performances and that PMT setting may be proposed as a source of variability in
MESF calculation that deserves a careful evaluation in quality-control trials.
Yamamura
Y, Rodriguez N, Schwartz A, Eylar E, Bagwell B, Yano N. A new flow
cytometric method for quantitative assessment of lymphocyte mitogenic
potentials. Cell Mol Biol (Noisy-Le-Grand) 1995;41 Suppl 1:S121-32
A new flow
cytometric method was developed to quantitatively assess lymphocyte
proliferation simultaneously for different subsets. The cells were stained with
a fluorescent dye, PKH-26 and were stimulated with mitogens. The fluorescence
intensities (FL2) of proliferating cells were measured by flow cytometry; and
each subset was identified by the use of a monoclonal antibody
(Mab)-fluorescein-isothiocyanate (FITC) (FL1). FL2 histograms were then
analyzed by the cell proliferation model based on the ModFit software (Verity).
This new method revealed information which could not be obtained by conventional
mitogen assays. For example, the CD4+ and the CD4- T-subsets responded to
phytohemagglutinin (PHA) quite differently from each other and it was indicated
that activation of one population could
significantly alter the response of the other. In addition, even
within a
subset, all activated cells did not proliferate uniformly. Some
cells
divided only once while others underwent further cellular division
during the
same time period. The method is, therefore, invaluable for
studying the
nature and the extent of interactions between different
cellular
subsets within a culture.
95298340
Zagursky RJ, Sharp D, Solomon KA
, Schwartz A. Quantitation of cellular receptors by a new
immunocytochemical flow cytometry technique. Biotechniques
1995 Mar;18(3):504-9
Flow cytometry
provides a rapid qualitative method for
analyzing
the binding of a fluorescent probe to a cell. To quantitate the binding
of a probe using flow cytometry, one must be able
to first calibrate the fluorescent signal with some known standard.
We have
compared a new one-step method with a previous two-step method
for
determining the number of binding sites (receptors) on the surface of
cells using
immunofluorescent staining of the cells and analysis by flow
cytometry.
Experimentally, recombinant chinese hamster
ovary cells,
expressing cell surface glycoprotein receptors, IIb/IIIa or Mac-1,
were assayed using specific mouse monoclonal antibodies against these
receptors. The two methods yielded comparable results and,
depending on
the cell type, detected anywhere from 100,000 to 300,000
antibody-binding
sites per cell, respectively.
95009206
Coder DM, Redelman D, Vogt RF. Computing
the central location of immunofluorescence distributions: logarithmic data
transformations are not always appropriate. [Review] [7
refs] Cytometry 1994 Jun
15;18(2):75-8
The
idea of the "average" intensity of immunofluorescence data is often poorly
defined, with such terms as average, mean, and peak used interchangeably.
In addition, the common use of logarithmic amplifiers with immunofluorescence
data further complicates the problem. Log amplifiers permit the display
of a wider range of fluorescence intensities. At the same time, they
effect a log transformation of the data. This transformation decreases
the variance resulting in narrower fluorescence distributions, which are
assumed to approximate normal distributions. When the log transform is
used, the distribution mean is the geometric mean of the untransformed data,
which is computed simply as the mean of the channel values. This mean
value serves as a simple indicator of the population center. Despite the
prevalence of log
transformations in flow cytometry, this transformation may not yield
normally distributed immunofluorescence data, whereas the square root or other fractional power transformations can yield normal distributions.
[References:
7]
94355905
Lanza
F, Rigolin GM, Moretti S, Latorraca A, Castoldi G.
Prognostic value of immunophenotypic characteristics of blast cells in
acute myeloid leukemia. Leuk
Lymphoma 1994;13 Suppl 1:81-5
In
order to elucidate the prognostic role of cytofluorimetry analysis of leukemic
cells in AML, the immunophenotypic characteristics of blast cells obtained from
66 AML patients belonging to M0-M2 and M4-M5 FAB subtypes have been investigated
by flow cytometry using a large panel of monoclonal antibodies (McAbs) utilized
in single, double, and triple fluorescence experiments. On a univariate
analysis, four different immunophenotypic blast cell characteristics were found
to be associated with a poor prognosis: expression of CD34 "bright"
(ratio > 10 between fluorescence emission of positive cells and that of
negative (isotypic) control-P/N ratio: mean MESF value: 265,000) in > 15%
blast cells, co-expression of CD34 and CD33 in > 60% blast cells, expression
of CD14 in > 30% leukemic cells, the MDR+ ("multiple drug
resistant") phenotype. In contrast, the duration of remission, and overall
survival of AML patients showing a "dim" CD34 expression (P/N ratio:
3-10: mean MESF value: 49,000) was similar to that of CD34- AML patients,
irrespective of the percentage of positivity for CD34, which was, however, a
predictive factor of survival in patients with higher CD34 fluorescence
intensities in their blastic population. No correlation between FAB subtypes,
prognosis and immunophenotype was found. The multivariate regression analysis
showed that, besides age, only the combined expression of CD34 and CD33 had
independent prognostic meaning. Indeed, in each FAB subtypes the CD34+/CD33+
phenotype was associated with a shorter survival and a lower mitotic rate. These
data may contribute to the understanding of the discrepancies so far observed in
the literature regarding the prognostic role played by the CD34 expression on
leukemic AML blasts.
95002942
Mainou-Fowler
T, Craig VA, Copplestone JA, Hamon MD, Prentice AG.
Interleukin-5 (IL-5) increases spontaneous apoptosis of B-cell chronic
lymphocytic leukemia cells in vitro independently of bcl-2 expression and is
inhibited by IL-4. Blood
1994;84(7):2297-304
During
hematopoiesis, viability factors that suppress apoptosis are required throughout
the differentiation process. Some of these factors may also function as growth
factors. Interleukin-5 (IL-5) is recognized as a growth factor in hematopoiesis.
We examined the involvement of IL-5 as a viability factor of B-CLL in vitro. In
13 B-CLL cases studied, IL-5 at 20 U/mL increased spo |