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Types of Flow 2

Brain Gym 2

A summary table-Flow systems

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PTO to find

Variability ofReaMetrixs Dry

Reagents

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ReaDigest1 0 o f D e c 2 0 0 9V o l u m e 1 , I s s u e 3

Focus of the month

Flow Cytometry

To know the answers forBrain Gym, log onto

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Merry Christmas

&

A Happy New Year

ReaMetrixs focus isthe development ofmedical diagnosticsolutions from aglobal perspective.

We intend to use theIndian Opportunityas an incubationcenter for thede ve l op m e nt ofproducts with an

international niche.

To learn more aboutour vision for thefuture of diagnostics,please visit our webpage: AffordableDiagnostics.

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ReaMetrix India Pvt. Ltd,50 B, 2nd Phase, TVSCross, Peenya IndustrialA r e a , P e e n y aBangalore- 560 058

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Phone: +91 80 2837

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Fax : +91 80 4117 2451

Flow cytometry History and Applications

Gary Salzman. In 1972, flow cytomters were entering the marketplacand Leonard Herzenberg (Stanforcame up with the term, FluorescenActivated Cell Sorter, or FACS. Flocytometers are very efficient, rapeasy to perform, less laborious, eato differentiate live and dead cetool when compared to the convetional tools like microscope, haemoctometer, microcyte and coultcounters.

Applications

Flow cytometry is a very useful toand is used worldwide. Its importanin scientific research and diagnosincludes detection of the presence specific surface and intracellulmarkers; measurement of metaboactivity and DNA content. The csorting applications of flow cytomter ranges from separation of larnumbers of cells for functional stuies or chromosomes for preparigene libraries to the direct cloning single rare transfected or hybridom

cells into each well of a tissue cultuplate. Flow cytometry finds applictions in the sectors of immunoflurescence, cell kinetics, cell cyckinetics, genetics, molecular biologanimal husbandry, microbiology, blogical oceanography, parasitologand bioterrorism.

History

Flow cytometry is the technologicalprocess for the individual measure-ments of cell fluorescence and lightscattering. This process is performedat rates of thousands of cells per sec-ond. This information can be used toindividually sort subpopulations ofcells. It is the measurement (meter) ofcharacteristics of single cells (cyto)suspended in a flowing saline stream. Abasic flow cytometer consists of 5main components- A light source

(Laser), a flow cell, optical componentstofocuslightofdif-

ferentcolorson tothedetectors, electronics to amplify andprocess the resulting signals and acomputer. Flow cytometry integrateselectronics, fluidics, computer, optics,

software and laser technologies ina single platform.

A long journey from microscopy

to FACS of today

The flow cytometer evolved fromthe development of several fields:-Microscopy, Dye chemistry, Elec-tronics and Computers. Withoutadvances in these fields it wouldnot have been possible to develop aflow cytometer. The real beginningof modern flow cytometry started

when Fulwyler (U.S. Los AlamosNational Laboratories) built a cellsorter using the Coulter principleto size cells and electrostaticcharging of droplets to sort them.

The development of the impulsecy-tophotometer (ICP) came next byDittrich and Gohde which wasknown as the phywe. Cells wereintroduced into a flowing sheathstream located under a high powermicroscope objective that providedthe optics for scatter measure-ments and fluorescence detection.By combining the measurement ofvolume, light scatter and fluores-cence into a single instrument, PaulMullaney (also at Los Alamos) in-troduced multiparameter flow cy-tometry. The ability to measureside scatter was added by exten-sive experiments carried out by

Do you know?

The first commercialized flow cytometric device was by a German developer and manufacturer Partecthrough Phywe AG in Gttingen in 1968-69.

The original name of the flow cytometry technology was pulse cytophotometry.

The first FACS instrument from Becton Dickinson came in 1974.

According to a market research report, the global flow cytometry market in 2008 stood at $1.5 billion andis estimated to grow to $3.7 billion by 2015 with estimated growth rates of existing product. About 68percent of the 2008 product revenue has been from instruments, and reagents make up 32 percent of therevenue.

The first fluorescence-based flow cytometry device (ICP 11) was developed in 1968 by Wolfgang Ghdefrom the University of Mnster, Germany (patent no. DE1815352)

Picture showing the scheme of optics and

illumination.Clinical microbiological reviews, Apr2000, p167-195

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Types of Flow Cytometers

Beckman Coulter. EPICS-XL is a singlelaser four color, six parametric opensystem. It utilizes Flow-Count Liquidbeads with premixed monoclonal anti-body combinations of three or fourantibodies.

Guava EasyCD4 volumetric system:The Guava EasyCD4 system (GuavaTechnologies) is a desktop modified flowcytometry based on a micro capillaryvolumetric cytometry technology. Itsnovel feature is that, it does not requiresheath fluid. The instrument uses twocolor monoclonal antibody combinationreagents and a volumetric control sys-tem that allows a precise count of cellnumbers and fluid volume, and is regu-lated by a variable-speed fluid (steppermotor syringe pump).

Partec CyFlow counter (volumetric

system): CyFlow is a desktop single-platform volumetric cytometry technol-ogy made by Partec, Germany. ThePartec flow cytometer is equipped withtwo sensing electrodes for a true volu-metric absolute counting with up to fivelight sources. It supports real timedata acquisition. CyFlow counter can beused as a mobile system that can run oncar batteries.

PointCARE system: This new flow sys-tem has been developed by PointcareTechnologies. The system is very com-

A brief of the commonly used flowcytometers based on microspherebeads and the volumetric method arementioned below.

FACSCount microbead-based sys-

tem: FACSCount system is the prod-

uct of Becton Dickinson Biosciences.It is the only available microbead-based single-platform instrumentthat is designed specifically for enu-merating the absolute CD4+, CD8+and CD3+ T-cell counts in no-lyse no-wash whole blood. It is a closed sys-tem.

FACScan/FACSCalibur instruments

with TruCount microbead-based

system: FACScan/FACSCalibur sys-tems are the product of Becton Dick-inson Biosciences. FACScan is a singlelaser three color five parametric opensystem and FACSCalibur is a duallaser four color six parametric opensystem. It utilizes TruCount absolutecount tubes (lyophilized pellet ofbeads in a test tube) or Liquid Tru-Count beads with premixed mono-clonal antibody combinations of threeor four antibodies to enumerateCD4+, CD8+ and CD3+ T-cell countsand/or Absolute lymphocyte counts.

EPICS-XL Instrument with Flow

count microbead-based system:EPICS-XL is an open system from

pact and portable and facilitates quickresults at the point-ofcare . It is aclosed system and used for CD4 enu-meration. The system requires minimaloperational training. It requires nocalibration and sample set up. It useslight scattering parameters and not

the fluorescence dyes to distinguishlymphocytes from white blood cells. Itis a mobile machine and can work onbattery back-up, however the sampleshave to be processed in a very shorttime (4 hours of collection).

LabNow CD4 system:

This portable system was developed byLabNow for rapid CD4 enumeration.The test is conducted in a single timeuse disposable microfluidics biochip inconjunction with a portable analyzer.The system facilitates minimal opera-tor interference that requires a singledrop of blood to be placed on to thecard for CD4 analysis.

Versatility of ReaMetrixs Dry reagents

The dry reagents are suitable to run on open flow cytometeras well as closed systems.

The dyes used in Dry Tri T-STATare qualified to assure supe-rior performance.

Innovative methodology by using ReaMetrix dry reagentsbrings about affordability of flow based testing by the optimi-zation of process involving reagent production without compro-mising on science.

Unitized single tube dried reagent can be used in all the flowsystems mentioned in the table alongside (except PointCAREsystem).

Brain Gym

Who am I?- Use the hints given below to find answers

1. I am an orange, fluorescent dye, have high quantumyield on binding to nucleic acids

2. I am a red fluorescent dye and I am a mixture of two

monosulfonyl chlorides (SO3 and SO2Cl)

3. I am a red protein and I bind the highest number ofPhycobilins (6 per alpha-beta subunit dimmer)

4. I am a blue dye from cyanobacteria with limited sup-pliers, I am an expensive dye

5. I am a fluorescent marker, adhere the GC-rich regionsof DNA, have a very large Stokes shift, test the cellviability

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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 novelplatform 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

System Features

BD FACSCount

system

Closed system - can only be used for limited applications.

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

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

4 decades of dynamic range

BD FACSCalibur

system

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

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

Volumetric system .

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

4 decades of dynamic range.

Guava PCA

system

Compact design.

Does not require Sheath Fluid to cause hydrodynamic focusing.

Open system.

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

4 decades of dynamic range.

Guava EasyCyte

Mini

system

Compact Design.

Does not require Sheath Fluid to cause hydrodynamic focusing.

Open system.

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

4 decades of dynamic range.

Summary of Flow Cytometric Systems

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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 amulti 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 withmulti-carousel loader.

Partec Cyflow SL

counter

Open system.

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

system

Closed system.

Automated gating.

Dedicated for CD4 and CD4%.

Provides 4 hematology parameters as well.

Primary vacutainer feed instrument.

Accuri C6

system

Open system.

Dual Laser (488, 635 nm), 4 color, two scatter detectors (FSC,SSC).

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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