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Focus of the month

ReaDigest Flow Cytometry

Wishing you all Vo l u m e 1 , I s s u e 3 1 0 o f D ec 2 0 0 9


“Merry Christmas

& Flow cytometry – History and Applications


A Happy New Year”
History software and laser technologies in Gary Salzman. In 1972, flow cytome-
a single platform. ters were entering the marketplace,
Flow cytometry is the technological
ReaMetrix’s focus is and Leonard Herzenberg (Stanford)
process for the individual measure- A long journey from microscopy
the development of came up with the term, Fluorescence
ments of cell fluorescence and light to FACS of today
medical diagnostic Activated Cell Sorter, or FACS. Flow
scattering. This process is performed
solutions from a The flow cytometer evolved from cytometers are very efficient, rapid,
at rates of thousands of cells per sec-
global perspective. the development of several fields:- easy to perform, less laborious, easy
ond. This information can be used to
Microscopy, Dye chemistry, Elec- to differentiate live and dead cells
We intend to use the individually sort subpopulations of
tronics and Computers. Without tool when compared to the conven-
“Indian Opportunity” cells. It is the measurement (meter) of
advances in these fields it would tional tools like microscope, haemocy-
as an incubation characteristics of single cells (cyto)
not have been possible to develop a tometer, microcyte and coulter
center for the suspended in a flowing saline stream. A
flow cytometer. The real beginning counters.
development of basic flow cytometer consists of 5
of modern flow cytometry started
products with an main components- A light source Applications
when Fulwyler (U.S. Los Alamos
international niche. (Laser), a flow cell, optical components
National Laboratories) built a cell Flow cytometry is a very useful tool
to
To learn more about sorter using the Coulter principle and is used worldwide. Its importance
focus
our vision for the to size cells and electrostatic in scientific research and diagnosis
light
future of diagnostics, charging of droplets to sort them. includes detection of the presence of
of
please visit our web specific surface and intracellular
dif- The development of the impulsecy-
page: “Affordable markers; measurement of metabolic
tophotometer (ICP) came next by
Diagnostics”. activity and DNA content. The cell
Dittrich and Gohde which was
sorting applications of flow cytome-
Contact us known as the “phywe”. Cells were
ter ranges from separation of large
introduced into a flowing sheath
ReaMetrix India Pvt. Ltd, numbers of cells for functional stud-
50 B, 2nd Phase, TVS stream located under a high power
ies or chromosomes for preparing
Cross, Peenya Industrial microscope objective that provided
gene libraries to the direct cloning of
A r e a , P e e n y a the optics for scatter measure-
Bangalore- 560 058 Picture showing the scheme of optics and single rare transfected or hybridoma
ferent illumination. ments and fluorescence detection.
India. cells into each well of a tissue culture
colors Clinical microbiological reviews, Apr By combining the measurement of
plate. Flow cytometry finds applica-
Phone: +91 80 2837
on to
2000, p167-195 volume, light scatter and fluores-
8693 tions in the sectors of immunofluo-
the cence into a single instrument, Paul
rescence, cell kinetics, cell cycle
Fax : +91 80 4117 2451
detectors, electronics to amplify and Mullaney (also at Los Alamos) in-
kinetics, genetics, molecular biology,
process the resulting signals and a troduced multiparameter flow cy-
animal husbandry, microbiology, bio-
computer. Flow cytometry integrates tometry. The ability to measure
logical oceanography, parasitology,
electronics, fluidics, computer, optics, side scatter was added by exten-
and bioterrorism.
sive experiments carried out by

PTO to find Do you know?


Types of Flow 2 •The first commercialized flow cytometric device was by a German developer and manufacturer Partec
Brain Gym 2 through Phywe AG in Göttingen in 1968-69.

Variability of 2 •The original name of the flow cytometry technology was pulse cytophotometry.
ReaMetrix’s Dry
Reagents •The first FACS instrument from Becton Dickinson came in 1974.
A summary table- 3-4 •According to a market research report, the global flow cytometry market in 2008 stood at $1.5 billion and
Flow systems is estimated to grow to $3.7 billion by 2015 with estimated growth rates of existing product. About 68
percent of the 2008 product revenue has been from instruments, and reagents make up 32 percent of the
revenue.
To know the answers for
•The first fluorescence-based flow cytometry device (ICP 11) was developed in 1968 by Wolfgang Göhde
‘Brain Gym’, log onto
from the University of Münster, Germany (patent no. DE1815352)
readigest.reametrix.com
1
Types of Flow Cytometers
A brief of the commonly used flow Beckman Coulter. EPICS-XL is a single pact and portable and facilitates quick
cytometers based on microsphere laser four color, six parametric open results at the point-of–care . It is a
beads and the volumetric method are system. It utilizes Flow-Count Liquid closed system and used for CD4 enu-
mentioned below. beads with premixed monoclonal anti- meration. The system requires minimal
body combinations of three or four operational training. It requires no
FACSCount microbead-based sys-
antibodies. calibration and sample set up. It uses
tem: FACSCount system is the prod-
light scattering parameters and not
uct of Becton Dickinson Biosciences. Guava EasyCD4 volumetric system:
the fluorescence dyes to distinguish
It is the only available microbead- The Guava EasyCD4 system (Guava
lymphocytes from white blood cells. It
based single-platform instrument Technologies) is a desktop modified flow
is a mobile machine and can work on
that is designed specifically for enu- cytometry based on a micro capillary
battery back-up, however the samples
merating the absolute CD4+, CD8+ volumetric cytometry technology. Its
have to be processed in a very short
and CD3+ T-cell counts in no-lyse no- novel feature is that, it does not require
time (4 hours of collection).
wash whole blood. It is a closed sys- sheath fluid. The instrument uses two
tem. color monoclonal antibody combination LabNow CD4 system:
reagents and a volumetric control sys-
FACScan/FACSCalibur instruments This portable system was developed by
tem that allows a precise count of cell
with TruCount microbead-based LabNow for rapid CD4 enumeration.
numbers and fluid volume, and is regu-
system: FACScan/FACSCalibur sys- The test is conducted in a ‘single time
lated by a variable-speed fluid (stepper
tems are the product of Becton Dick- use’ disposable microfluidics biochip in
motor syringe pump).
inson Biosciences. FACScan is a single conjunction with a portable analyzer.
laser three color five parametric open Partec CyFlow counter (volumetric The system facilitates minimal opera-
system and FACSCalibur is a dual system): CyFlow is a desktop single- tor interference that requires a single
laser four color six parametric open platform volumetric cytometry technol- drop of blood to be placed on to the
system. It utilizes TruCount absolute ogy made by Partec, Germany. The card for CD4 analysis.
count tubes (lyophilized pellet of Partec flow cytometer is equipped with
beads in a test tube) or Liquid Tru- two sensing electrodes for a true volu-
Count beads with premixed mono- metric absolute counting with up to five
clonal antibody combinations of three light sources. It supports real time
or four antibodies to enumerate data acquisition. CyFlow counter can be
CD4+, CD8+ and CD3+ T-cell counts used as a mobile system that can run on
and/or Absolute lymphocyte counts. car batteries.
EPICS-XL Instrument with Flow PointCARE system: This new flow sys-
count microbead-based system: tem has been developed by Pointcare
EPICS-XL is an open system from Technologies. The system is very com-

Versatility of ReaMetrix’s Dry reagents Brain Gym


The dry reagents are suitable to run on open flow cytometer “Who am I?”- Use the hints given below to find answers
as well as closed systems.
The dyes used in Dry Tri T-STAT are qualified to assure supe-
1. I am an orange, fluorescent dye, have high quantum
rior performance.
yield on binding to nucleic acids
Innovative methodology by using ReaMetrix dry reagents 2. I am a red fluorescent dye and I am a mixture of two
brings about affordability of flow based testing by the optimi-
monosulfonyl chlorides (SO3 and SO2Cl)
zation of process involving reagent production without compro-
mising on science. 3. I am a red protein and I bind the highest number of
Phycobilins (6 per alpha-beta subunit dimmer)
Unitized single tube dried reagent can be used in all the flow
systems mentioned in the table alongside (except PointCARE 4. I am a blue dye from cyanobacteria with limited sup-
system). pliers, I am an expensive dye
5. I am a fluorescent marker, adhere the GC-rich regions
of DNA, have a very large Stoke’s shift, test the cell
viability

2 We welcome your feedback, write to us at readigest@reametrix.com


Industry New(s)
New flow cytometry platform used for variety of clinical diagnostic assays– Accuri C6
Accuri is a bench-top, dual laser four color six parametric open system. It is a new genera-
tion flow cytometer that would require minimal training and operator expertise. Its novel
platform does away with pre acquisition compensation and
operates with newer technology and minimal settings. Ac-
curi is the only flow based system that can perform volu-
metric and micro bead based assays.

For more information visit


www..accuricytometers.com

Summary of Flow Cytometric Systems

System Features

BD FACSCount Closed system - can only be used for limited applications.

Requires minimal operator interaction in acquisition and analysis, automated gating.


system
Single laser (532 nm), 2 color, single scatter detector (FSC).

4 decades of dynamic range

BD FACSCalibur Open system with optional MultiSet software for automated gating of some assays.

Optional FACSLoader system allows for automated analysis of nearly 40 tubes.


system
Volumetric system .

Dual laser (488 nm and optional 635 nm), 2 scatter detectors (FSC/SSC).

4 decades of dynamic range.

Guava PCA Compact design.

Does not require Sheath Fluid to cause hydrodynamic focusing.


system
Open system.

Single laser (532 nm), 2 color, single scatter detector (FSC).


4 decades of dynamic range.

Guava EasyCyte Compact Design.


Mini Does not require Sheath Fluid to cause hydrodynamic focusing.

system Open system.

Single laser (488 nm), 3 color, two scatter detectors (FSC,SSC).


4 decades of dynamic range.

Continued on P4...

3
Summary of Flow Cytometric Systems (continued from p3)

System Features

Coulter EPICS XL Open system.

Single Laser (488 nm), 4 color, two scatter detectors (FSC,SSC).


4 decades of dynamic range.
CoulterPrep Station automation option available for sample preparation, together with a
multi carousel loader.

Coulter FC500 Open system.

Dual Laser (488, 635 nm), 5 color, two scatter detectors (FSC,SSC).
20 bit system with digital post acquisition compensation.
CoulterPrep Station automation option available for sample preparation, together with
multi-carousel loader.

Partec Cyflow SL Open system.


counter Single Laser (488 nm), 3 color, 2 scatter (FSC,SSC).
Sub-micron scatter size resolution.

Volumetric system.
16 bit system.

Can be 12 V battery operated.

PointCARE Closed system.

Automated gating.
system
Dedicated for CD4 and CD4%.

Provides 4 hematology parameters as well.


Primary vacutainer feed instrument.

Accuri C6 Open system.

Dual Laser (488, 635 nm), 4 color, two scatter detectors (FSC,SSC).
system
24 bit system with digital post acquisition compensation.
Gainless operation - factory set gains, enabling simple user interaction with the instru-
ment.
Accommodates variety of inputs - 96 well plate, FACSTubes, eppendorfs.

Non-pressurized fluidics ensures minimal consumption of sheath.

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