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Concepts and Developments in Flow Cytometry and Cytomics at
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Cytophotometry
(1) and
electronic cell counting
(2,
3)
generated from early on in the history of flow cytometry a
significant interest amongst biomedically oriented scientists.
It took, however, a certain time until eminent clinical hematologists were
convinced of the usefulness of electronic red and white blood cell and platelet
enumeration by Coulter counters
(4,
5,
6)
in comparison to the long established counting chamber methods.
To speed up the acceptance process, Wallace Coulter as globally
thinking entrepreneur and instrument developper travelled around 1958/59
personnally with a Coulter Model A counter in his luggage to various
institutions in Europe that he considered of strategic interest. One of
these institutions was the
Max-Planck-Institute für Biochemie in Munich (MPI-Biochemie), headed by nobel laureate
Adolf Butenandt
where he addressed Gerhard Ruhenstroth-Bauer, a hematology oriented
scientist and director of the Department of Experimental Medicine.
Ruhenstroth was interested, bought a Coulter A counter with a serial
number around 550 and the particular interest to measure volume distribution
curves of cells. This is possible because the signal amplitude of the counting
pulses in electrical cell sizing is proportional to cell volume. He
considered this feature of the instrumentat of particular interest for the
better characterisation of cells from blood, leukaemias and cancers.
Thus Klaus and later Odila Zang, two young scientists of his laboratory, investigated volume distribution curves of various cell types (7) and recognized that Coulter cell volume distributions were right skewed for erythrocytes. This was considered of probably artefactual nature since from previous microscopic and electron microscopic evidence, symmetric Gaussian normal distribution curves were expected. Wallace Coulter being primarily interested in the use of electronic blood cell counting for clinical purposes was not enthousiastic about these unforeseen findings and considered legal action against the MPI-Biochemie for distributing non advantageous rumours about his counter.
Ruhenstroth, remaining nevertheless highly interested in the determination of cell volume distribution curves, circumvented potential conflicts with Coulter by asking Butenandt for an equivalent of 250.000 Euro (countervalue of 10 high speed ultracentrifuges at this time, for example of Beckman Spinco L-50) to develop new instrumentation. Butenandt was hesitant but finally made these comparatively enormeous funds available. Jürgen Gutmann was hired as electronic engineer, an electronic as well as a workshop were equipped and the instrument building phase of the experimental medicine department began (8 --> fig.1, 9, 10, 11 --> fig.2 left) in its former location in central Munich, close to the main railway station (Goethestrasse 31) from where the institute moved to the newly built Martinsried facilities in 1972.
fig.1 Electrical impedance cell sizing unit, J Gutmann, Diplomarbeit Fachbereich Elektrotechnik TU-München 1963 |
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fig.2 left: Signal processing & histogram display unit from: J Gutmann, V Kachel, R Röttger, G Ruhenstroth-Bauer, Naturwiss 55:130(1968) (reproduction with kind permission of © Springer-Verlag Berlin Heidelberg), right: Metricell, Dec 1971 (image: © Günter Valet) |
Klaus & Odila Zang, Jürgen Gutmann, Mac Fulwyler (12) and others observed the right skew of Coulter volume distribution curves, all considering it an artefact. Gutmann hypothesized that biconcave erythrocytes went through the sizing orifice in variable orientation like lengthwise, transverse or in intermediate position causing variable displacement of electrical field lines leading to increased signals for transversely passing erythrocytes. Right skew in nucleated cell volume distribution curves was interpreted as representing in part size difference during cell cycle.
Gutmann's concept was not confirmed by the subsequent investigations initiated by Reinhard Thom, a clinician from Berlin (Klinikum Westend, Freie Universität Berlin). He modified a standard Coulter orifice by a hydrodynamically focused cell influx capillary in front of the sizing orifice while being a guest scientist at the MPI-Biochemie (13, 14, 15). The available Metricell sizing instrumentation, as developed by Volker Kachel (16) (fig.2 right) in continuation of Gutmann's earlier work was used for the measurements.
The interest in path, shape and orientation of the cells on the way
through the orifice required their photographic visualization,
using a special observation chamber in combination with ultrashort flashlight (40nsec) pulses
(16 -> fig.4a).
A quartz observation chamber had been fabricated earlier by Zeiss (Oberkochem)
during a collaborative effort 1966/67 that had been stimulated by the
development of the first optical flow cytometer in a modern sense by
Kamentsky
(17). In this cytometer, cell DNA was determined
by optical absorption at 253.7nm simultaneously with a cell light scatter
measurement at 410.0nm. The project idea with Zeiss was to simultaneously
measure DNA at 253.7nm and protein content at 280nm together with the
electricaly determined cell volume.
Zeiss provided a tuneable monochromator with HBO200 mercury arc lamp
in conjunction with a sophisticated UV-microscope. The
measurements were technically possible with this setup but it became
soon clear that the observed signals were mostly light scatter signals
and therefore not sufficiently specific for the separation between
DNA and protein of unstained cells in flow.
The project was abandoned but the quartz
chamber, kindly donated by Zeiss, proved very useful for the subsequent
extensive high speed photographic investigations of cells passing through
electrical sizing orifices.
The photographs showed that cells travelled always lenghthwise in the
quickly accelerating fluid stream through the orifice.
Cell passing over the edges of the orifice entrance, as possible in
the typical Coulter counter, passed, however, through zones of elevated
electrical field strength, resulting in higher electrical pulse signals
than cells passing through the center of the orifice. Focusing particles
on restricted pathways through the orifice provided similar electrical
field strength conditions for all measured cells, resulting in the
postulated symmetrical volume distribution curves for erythrocytes
(14,
18)
or monodisperse microbeads.
Spielman & Goren
(19)
had equally observed a narrowing of Coulter volume distribution curves by
hydrodynamic focusing around the same time but did not provide
experimental explanations for their observation.
Conclusion: The extensive experimental work connected with the proof
of the hypothesis that the right skew of volume distribution curves in normal
Coulter counters at unrestricted particle path through the sizing orifice
was an artefact has lead to the development of the cell sorter
(12)
as well as to fast imaging in flow
(20)
A major bottleneck in cytometric investigations concerned from the beginning on
data display and data analysis, given the comparatively high data
acquisition rates between typically 1.000 and 5.000cells/sec.
The initial hardware solution at MPI-Biochemie consisted of a
set of around 20 relays having little weights attached to strings running
over small step wheels at each relay. Each wheel was advanced by its relay
after a given number of counted cells within its window of pulse
amplitudes (channel) that is within a certain cell size range. This provided "negative cell
size histograms" (fig.2 left).
The absolute cell count for each histogram channel was separately printed
on paper. The use of an oscilloscope as histogram display and of an
xy-plotter to permanently visualize the measured histograms
(fig.2 right) improved record keeping but the quantitative information
for histogram comparison and further analysis remained still printed on paper.
The increased resolution of hydrodynamically focused measurements of cell
volume distribution curves lead to the discovery of discrete erythrocyte
populations by size during the first trimenon of life
(21)
in various mammalian organisms or in adults after x-irradiation
(22)
or strong bleeding. Depending on species, these populations showed in
addition different hemoglobins, antigen expression or electrophoretic
mobility as consequence of switched gene expressions patterns.
Having shown that the volume distribution curves had the potential to
monitor gene activation patterns in the hematopoietic system, a more
detailed data analysis was required to understand the sequence of events.
The printed cell contents of the various channels of the volume distribution
histograms were analysed in a first approximation on probability paper
(21,
22)
to obtain a model of the sequence of cell populations, thus concentrating
the many initial histogram channel counts into a sequence of means, standard
deviations and % contribution of the various cell populations over time.
Computers were at this time far too expensive to purchase them for such an
analysis. The move of the institute to Martinsried in 1972 provided,
however, access to a Siemens 4004 main frame computer that had been
purchased by the Max-Planck Gesellschaft for the equivalent of around
2 million Euros to advance the evaluation of electron microscope and x-ray
crystallography data. The computer had a core memory of around 1Mbyte.
From now on, cell volume distribution curves could be iteratively fitted
by standard Gaussian normal distributions or by other functions with
substantially more information and knowledge being extracted
(23,
24)
than by the visual inspection of histograms. It was also easier to
evaluate cell aggregation for example of erythrocytes as cause of the clinically
observed erythrocyte sedimentation rate (ESR) in anticoagulated blood samples
(23).
fig.3 Fluvo Metricell 1982, Günter Valet, Hanna Kahle, (reproduction with kind permission of © KNA Bild, Bonn) |
With the introduction of fluorescence into flow cytometry by Wolfgang Göhde (25, 26) and into cell sorting in Len Herzenberg's laboratory (27), flow cytometry gained access to an essentially unlimited number of specific molecular stains. It seemed especially important for medicine oriented cell biochemistry to develop into this direction since work prior to the development of fluorescence flow cytometry had shown the importance of relating biochemical changes in tissues to the cellular level in order to better understand their biological and physiological significance (28). Phywe company (Göttingen) commercialized Göhde's instrumentation but difficulties existed in purchasing only the optical part of the instrument since the intention at MPI-Biochemie was to use own electronics for signal amplification and list mode data acquisition as well as a performant computer for data evaluation. A member of the Phywe board of directors happened to be senator of the Max-Planck-Gesellschaft. He arranged the purchase of the optical part of the first commercially produced Phywe ICP11 instrument (W Göhde personal communication) in 1969. |
Dr.Mildred Scheel, the wife of former German Bundespräsident Walter Scheel had founded the Deutsche Krebshilfe as well as the Mildred-Scheel Foundation to provide better care for cancer patients and to advance cancer research in Germany. Following significant funding of clinical institutions, certain criticisms emerged that not enough was done for the research sector. This prompted the Mildred-Scheel Foundation together with the Max-Planck Gesellschaft zur Förderung der Wissenschaften to internationally announce a 5 year research project in the area of medicine oriented basic cancer research. Günter Valet applied as did more than 50 other scientists and was finally appointed head of the independent Mildred-Scheel Laboratory for Cancer Cell Research (fig.4) after a thorough two year selection process by a search committee, consisting of around 20 eminent scientists from the Deutsche Krebshilfe, the Max-Planck-Gesellschaft as well as from several universities and cancer research institutes in Germany.
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fig.4 Opening of the Mildred-Scheel Laboratory for Cancer Cell Research at Max-Planck Institut für Biochemie, Martinsried on July 15, 1981, Dr. Mildred Scheel (President Deutsche Krebshilfe), Prof. Peter Hans Hofschneider (Institute Director) (reproduction with kind permission of © MPG-Pressestelle, Munich) |
The submitted project proposal aimed at the simultaneous multiparameter
analysis of single cells by flow cytometry as a sensitive approach for
the automated detection and molecular characterization of cancer cells in
patients as well as at the development of suitable benchtop instrumentation
for this purpose. The Mildred-Scheel Foundation provided the
equivalent of 1.5 Mio Euro for salaries while the Max-Planck Gesellschaft
contributed the laboratories and running costs in the order of 0.5 Mio Euro.
Dr.Scheel as former radiologist, followed the project with
close attention but died in 1985 of cancer.
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fig.5 Cell function in cytomics: left: intracellular pH & esterase staining (ADB/DCH) of viable rat bone marrow cells (blue) and of the DNA in dead cells (PI) (red). right: contour line display of the flow cytometric measurement of around 6.000 cells of the stained cell preparation from the left panel. |
Major focus points of the scientific project work concerned the:
- establishment of sensitive
cell function assays
for cancer cell detection by flow cytometry (fig.5)
- development of individualized
cytostatic drug assays
for patient cells
- development of data pattern
analysis
for knowledge extraction from complex multiparameter data
- application of this new potential to
patient studies
in collaboration with a variety of clinical institutions
- development of benchtop instrumentation (FLUVO-Metricell II, Cytomic123,
34,
35,
36,
37)
by Volker Kachel
for further details of the cell biochemistry group see
literature references
1981-1990.
After 5 years, the project was prolonged for another 3 years until 1989,
amounting to a total funding of around 3.2 Mio Euro. It was at this time the
largest project, the foundation had ever funded. About 10 FLUVO-Metricell II
instruments, data recording or display modules were produced by the technical
group and sold to scientific institutions between 1984-1989 with reinvestment
of the income into further research. The reasons for the termination of
the project in 1989 were that flow cytometry had become a routine technology
and research in the earlier context was not considered of primary interest any more.
Following the termination of the Mildred-Scheel Laboratory project,
the cell biochemistry part of the Martinsried flow cytometry group
remained as Cell Biochemistry Group scientifically independent and continued
the work with funds from European research projects, through
Sonderforschungsbereiche of the Deutsche Forschungsgemeinschaft
(DFG) and basic funding by the Max-Planck-Gesellschaft.
Given the fast technological progress, commercial instrumentation such as the
PASIII cell analysis and closed piezo cell sorter system (Partec, Münster, Germany)
met increasingly the needs of basic cell biochemistry research and did not
require further in-house technological development. Likewise, personal
computers provided enough computing power for cytometric list mode data
analysis.
Decreasing support in combination with an increasing accumulation of
multiparametric data sets in many clinical environments generated a
gradual shift from the production of own data to processing other
groups data. This was advantageous since it would have been
experimentally very difficult to generate the amount of experimental
and clinical data required for the development of these concepts within a
single laboratory.
The limited interest for flow
cytometric cell biochemistry at the national level (see frequency of terms
like Zytomik or Humanzytomprojekt / Humanzytom-Projekt on the
Internet) favoured this increased concentration onto the intellectual core
competence of knowledge extraction from complex multiparameter data
at the international level. Essential transit points in this effort over time were:
- the
automated diagnosis
from flow cytometric list mode data (1987)
- the view that cytometry and later
cytomics
constitute a
biomedical
key discipline
where the explicit analysis of the heterogeneity of cell systems in
form of
system
cytometry
(1997) represents a comparatively efficient top-down strategy for
the systematic resolution of the cellular and molecular biocomplexity of
higher organisms with one of the important advantages being that complex disease
mechanisms can be efficiently investigated without necessity for extensive a-priori
knowledge and molecular pathway modeling.
- the individual patient
disease
course prediction by SMDC
(predictive medicine by SMDC) (2000) (fig.6)
- the definition of cell systems as
cytomes
(2001) in combination with
- the introduction and redefinition of the plant science term
cytomics for
cell biochemical purposes
- the
predictive medicine
by cytomics concept (2001) as well as
- the elaboration of concepts for a
human cytome project
(2004) and for a
periodic system of cells
(2005)
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fig.6 CLASSIF1 data pattern analysis: Discriminatory disease classification masks (top of rightmost column) consisting in this case of the 5 most discriminatory out of 44 measured thrombocyte parameters, permit to correctly distinguish between risk and non-risk patients for myocardial infarction from molecular properties of peripheral blood thrombocytes. The CLASSIF1 algorithm (CLASSIF1 classification column) correctly recognizes the two types of patients (clinical classification column) from the analysed flow cytometric data (details, 38) |
Excellent collaborators in the cell biochemistry and technology areas as well as a significant number of highly interested clinicians have very much contributed to the success of the various scientific projects. I want to thank them all for their continued enthousiasm and their committment to the goals of this highly transdisciplinary work. I am furthermore very grateful to several scientists at the national and international level for their openness to the conceptual aspects of this work. They have by numerous discussions and a number of joint publications very significantly contributed to the elaboration and dissemination of the cytomics, predictive medicine by cytomics and human cytome project concepts
| Cell Biochemistry | Technology | Clinicians | Concepts |
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Gregor Rothe Sven Klingel Andreas Oser Michael Collasius Christoph Zirkelbach Jeanette Malin-Berdel Alexander Raffael Lorenz Rüssmann Vincentiu Manta Max Hasmann Gerburg Wulf Susanne Burow Jürgen Treumer Hella Horst Ganesh Shankar Ken Trevorrow Tarek Elsherif Hanna Kahle |
Volker Kachel Eberhard Menke Gerhard Benker Karl Schedler Heinrich Schneider Ernst Kordwig Ewald Glossner |
Friedrich Otto Scheyffarth Rainer Wirsching Florian Liewald Hans Heinrich Warnecke Wolfgang Kellermann Thomas P.U. Wustrow Rolf Lamerz Hansjörg Sauer Diethelm Tschöpe Heinz Gerd Höffkes Andreas Neubauer Herbert Leyh Rainer Repp Thomas Dörner Luc Kestens Jan Gratama Elisabeth Bräutigam |
Attila Tarnok Paul Robinson Enrique O'Connor Andreas Radbruch Peter van Osta Bob Murphy Andres Kriete Gerd Schmitz Gero Brockhoff Susann Müller |
The Martinsried flow cytometry group in its cell biochemistry and technology
branches, has contributed to several developments and concepts that have
significantly shaped the cytometry field over time:
1. The early instrument development terminated the long
going controversy about the right skewedness of cell volume distribution
curves by electrical sizing by recognizing the importance of hydrodynamic
focusing. It furthermore provided early computerized list mode data
acquisition as an important prerequisite for the efficient extraction of
information and knowledge from multiparameter flow cytometry measurements.
2. The development of flow cytometric
cell function assays
enabled the fast molecular evaluation of disease states as well as predictions
about the future disease progress in individual patients. The flow cytometric
determination of cell function has advanced on its own into a
steadily growing field in medicine and cell biochemistry but also in the
pharmaceutical industry in form of high-throughput and high-content
assays for drug discovery, both by flow cytometry and image cytometry.
3. The development of data pattern analysis (data sieving) by the
CLASSIF1 algorithm
permits the exhaustive and standardized knowledge extraction from flow
cytometric list mode files as well as from multitudes of other multiparameter
data.
4. Data pattern analysis enabled amongst others the development of the concepts of
individual patient disease course prediction by SMDC
(standardized multiparameter data classification) and
system cytometry
as well as the
cytome & cytomics
definitions
(Omics-glossary
(2001)),
leading to the concepts of
predictive medicine by cytomics,
of a
human cytome project
and of a
periodic system of cells
by cytomics.
The experimental and conceptual work of the Martinsried Cell Biochemistry
Group has led to diverse forms of
external interest
such as the election into leading positions of international scientific
societies, membership in various editorial boards, associate editor
& editor of the "cytomics" editorial column in Cytometry A, invitation to more than 20
review articles in the cytomics area during the time period 2001-2006 and to
more than 200 invitations for presentations of the groups cell biochemistry
work at scientific meetings or in many
institutions worldwide since 1981, furthermore to awards, membership in
scientific advisory boards and collaboration with the pharmaceutical industry
in the area of predictive medicine by cytomics (-> personalized medicine,
individualized medicine). The presently widespread international interest
is also reflected by inclusion of various definitions and concepts into
on-line glossaries
like Omes & Omics
or enzyclopedias like Wikipedia
(cell biochemistry,
predictive medicine,
cytomics,
human cytome project)
or in the ranking of the groups Internet pages by search engines like
Google (date: position/total number of hits):
| cell function, "cell function" | cell biochemistry, "cell biochemistry" | predictive medicine, "predictive medicine" | cytome(s) | cytomics | human cytome project |
21.10.07: 10/186mio, "2/2.0mio" 06.04.08: 2/20.5mio, "2/1.2mio" |
18.11.04: 5/75.400 14.04.07: 4/42.4mio,"3/161.000" 06.04.08: 3/3.5mio,"2/60.400" |
02.08.01: 32/43.000 26.02.03: 5/75.400 14.04.07: 4/1.69mio,"4/88.100" 06.04.08: 5/388.00,"5/42.100" |
02.08.01: --/-- (0/0)
10.08.01: --/-- (1/3) 26.02.03: --/-- (1/18) 14.04.07: 3/88.900 (1/337) 06.04.08: 4/28.100 (8/542) |
02.08.01: --/81
05.04.02: 1/150 07.09.05: 6/9.730 14.04.07: 3/40.600 06.04.08: 3/20.000 |
12.12.03: --/3
16.12.03: 3/10 06.04.05: 1/139 14.04.07: 3/14.000 06.04.08: 3/10.400 |
The fields of cytometry and
cytomics
have been fascinating over many years by the collaboration of scientists
being interested in trans-disciplinary
(cross-disciplinary) concepts and their potential to provide
entirely new insights into cellular biomechanisms. Flow cytometry and image
cytometry instrument developpers were initially (1965-1985) rather competing
against each other for supremacy in the sensitive detection of cancer cells
but the efforts have become more and more collaborative and mutually
complementing in recent years. Flow cytometry with its early on fluorescence orientation
has substantially enhanced the development of new stains,
proving equally useful for molecular biology as for single cell oriented
image cytometry like in confocal or laser scanning microscopy.
The cytometric field continues to be fascinating through its
potential to unfold the organismal (organismic) biocomplexity
top-down by single cell molecular analysis in-situ that is with all molecules
in place, lending itself to the reverse engineering of the assembled molecular
machinery as well as to the investigation of the natural heterogeneity of
cells in tissues and in complex disease mechanisms as one of the bases for
the adaptivity of organisms in variable environments. The top-down approach
seems particularly promising since the knowledge of the entire set of
biomolecules as derived at the genome level does by itself so far not
provide enough information for their molecular reassembly in form of living
cells, tissues, organs or organisms.
The fascination for the cell biochemistry and cytomics fields will therefore
in all likelihood not only continue but further increase, seen the overall
potential and challenges of this approach.
Download the ZIP file containing all Cell Biochemistry pages for example into directory: d:\classimed\, unzip into the same directory, enter the address: file:///d:/classimed/cellbio.html into the URL field of the Internet browser to directly access text & figures on your harddisk free of network delays (further information).
| © 2010 G.Valet |