coulter counter

Coulter Counter

Physiology Seminar

Mrinmoy Pal

Graham, Marshall Don. "The Coulter principle: foundation of an industry." Journal of the Association for Laboratory

Automation 8.6 (2003): 72-81.

Wallace H. Coulter Joseph R. Coulter

Wallace H Coulter discovered the Coulter Principle in the late 1940s (though a patent was not awarded until October 20, 1953). Coulter was influenced by

the atomic bombs dropped on Hiroshima and Nagasaki. These events motivated Coulter to simplify and improve blood cell analysis so that large

populations could be screened rapidly, as would be necessary in the event of a nuclear war. Remembering his visits to hospitals, where he observed lab

workers hunched over microscopes manually counting blood cells smeared on glass, Wallace focused the first application on counting red blood cells.

1935, he joined General Electric X-Ray as a sales and service engineer, electronics companies

Coulter Counter

Coulter counter is commercially available device for

determining the size distribution of electrically

nonconducting particles suspended in a conducting medium.

Two electrodes passing constant current are placed on

either side of a small hole or aperture through which the

suspension is sucked. Because of the smallness of the

aperture the major resistance in the circuit is at the aperture

and when a nonconducting particle passes through , the

resistance is changed giving rise to electrical pulse.

. The number of pulses is equal to the number of cells

counted and the strength of the signal (pulse height) is

directly proportional to the cell volume.

The electrical response of the instrument is essentially

independent of the shape of particles with the same volume,

an exception to this may occur with some extreme shapes.

Color or refractive index of the particles does not affect the

results.

Coulter, Wallace H. "High speed automatic blood cell counter and cell size analyzer." Proc Natl Electron Conf. Vol. 12. 1956.


Automation 8.6 (2003): 72-81.

Working Principle


The pulses produced at the orifice are amplified and displayed on the oscilloscope screen and appear as vertical lines or spikes. The height of an individual pulse spike from the baseline is a measure of relative size of the cell. The threshold control dial enables the user to set a cut off. Only if the pulse exceeds the threshold,

it gets counted. In addition to a display of relative cell size the oscilloscope also indicates the threshold control by brightening that portion above the threshold level .

Electronic System & Output

The Model A Coulter CounterThe round black object at the upper rightof the stand is the stirrer motor used tokeep heavy particles in suspension. Theconsole contains, from left to right, themechanical totalizer used for slowlyaccumulating high-value digits, the threedecade counters used for the rapidlyaccumulating low-value digits, and theoscilloscope display tube. Controls for thesingle threshold and the aperture current

appear below the display.


Major Drawbacks Single threshold: Multiple sample runs at successively increasing thresholds were requiredfor generating a cumulative size distribution. The voltage source: Accurate counting and sizing was hampered by instrument’s sensitivityto aperture dimension and temperature-induced resistivity change of the electrolyte.

Commercial Models

The Model B Coulter Counter FN Model

Commercial Models

Improvements:Dual-threshold current-sensitive amplifierMulti-channel Pulse Height Analyzers


Automation 8.6 (2003): 72-81.

Experimental Considerations

• Coincidence:

Anomalous electrical pulses can be generated when multiple particlesenter the aperture simultaneously. This situation is known ascoincidence. This occurs because there is no way to ensure that a single largepulse is the result of a single large particle or multiple small particlesentering the aperture at once. To prevent this situation, samples must be fairlydilute.

Primary Coincidence Secondary Coincidence


• Porous particles

The Coulter Principle measures the volume of an object, since thedisturbance in the electric field is proportional to the volume ofelectrolyte displaced from the aperture. This leads to some confusionamongst those who are used to optical measurements from microscopes orother systems that only view two dimensions and also show the boundaries ofan object. For porous particles the volume of pores that are parallel to theelectric field lines is not measured, while the volume of pores perpendicularto these lines does contribute to the measured volume. Thus, Particles withinterconnected pores produce a size related to their solid volume as shownbelow.

Envelope volume

Solidvolume

Porous particle


• Particle path

The shape of the generated electrical pulse varies with the particle paththrough the aperture. The use of the Edit facility (signal processingalgorithm) eliminates the distorted pulses, so that the recovered sizedistribution is close to the correct one. Using a longer tunnel aperturemay enhance the accuracy of the results for samples with a narrow sizedistribution. This behaviour of long tunnel apertures is mostly due to thegreater approach to parabolic flow inside the bore. Still better engineeringtechnique was required to correct for artifacts resulting from particle path.

The method consists of feeding the suspension from a 30 μ diameter hole positioned 2 to 4 mm from the aperture of the Coulter device. Both tubes are immersed in a bath of particle-free electrolyte.

When the electrolyte is sucked through the sensing aperture, the stream tube containing the suspension is accelerated and thinned so that the particles all travel essentially the same

streamline, and consequently have uniformity of approach and passage through the sensing aperture.

Spielman, Lloyd, and Simon L. Goren. "Improving resolution in Coulter counting by hydrodynamic focusing." Journal of Colloid and Interface Science 26.2 (1968): 175-182.

Hydrodynamic Focusing

Sweet, R.G. 1965. High frequency recording with electrostatically deflected ink jets. Rev. Sci. Instr. 36: 131–136.

Richard Sweet

RG Sweet’s Ink Jet ‘‘Oscillograph’’ which was the basis for the oscillation for separating cells into single droplets in Fulwyler’s cell sorter.

Cell Sorter

Fulwyler, Mack J. "Electronic separation of biological cells by volume." Science 150.3698 (1965): 910-911.

Mack Fulwyler

Mack Fulwyler used Wallace Coulter’s Coulter principle as the basis for identifying different red blood cells in his cell sorter invention.

Cell Sorter

Cell counting and sizing: A cell suspension enters the dropletgenerator (C) by way of a tube (D) and emerges as a high-velocity fluid jet (E). Cell volume is sensed as the cell passesthrough a Coulter aperture within the droplet generator (C). Anelectric pulse proportional to cell volume is obtained at J.

Droplet formation: A piezoelectric crystal (A), driven at afrequency of 72,000 cy/sec, produces vibrations which passdown the rod (B) into the liquid within the droplet generator.The shape of the rod serves to amplify the magnitude of thevibrations within the liquid. The velocity fluctuations of theemerging liquid produce bunching of the liquid column.

Cell Sorting: Now the isolated cell droplet arrives at theseparation point (I). The size of the charging pulse needed todeflect droplets into the proper vessel is electronicallydetermined from the cell-volume pulse and the droplet ischarged electronically at the charging collar F. The chargeddroplets are then deflected (H) on entering the electrostaticfield between the deflection plates (G). Finally, a series ofcollection vessels (L) receive the deflected droplets.

Fulwyler, Mack J. "Electronic separation of biological cells by volume." Science 150.3698 (1965): 910-911.

Cell Sorter

FACS: Fluorescence-activated cell sorting

Becton Dickinson (1974) Source: http://docs.abcam.com/pdf/protocols/Introduction_to_flow_cytometry_May_10.pdf

The Flow system (fluidics) The Optical system (light sensing)

Once the sheath fluid is running at laminar flow, the cells are injected into the center of the stream, at a slightly higher pressure. The principles of hydrodynamic focusing cause the cells to align in a single file.

Advancement in technologies such as fluorescence tagging and light scattering

The intensity of fs signal has been attributed to cell size and the intensity of ss signal is proportional to the amount of cytosolic structure in the cell.

FACS: Fluorescence-activated cell sorting

Source: http://docs.abcam.com/pdf/protocols/Introduction_to_flow_cytometry_May_10.pdf

Application:Immunophenotyping, DNA Ploidy and S-phase Fraction Analysis

The Electronic system (signal processing)

Lab-on-a-chip Coulter Counter

Mei, Zhe, et al. "Counting leukocytes from whole blood using a lab-on-a-chip Coulter counter." 2012 Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE, 2012.

A lab-on-a-chip Coulter counter allows cancer patients going through chemotherapy and patients with

compromised immune systems to take less than 10uL of blood to measure lymphocyte concentration. The test can be self-administered or performed at the point-of-care clinics to reduce costs and risks of hospital infection. Instead of electroplated or deposited metal electrodes, off-the-shelf gold pins were used as

electrodes to simplify fabrication process and to achieve superior uniformity in E-field distribution for improved signal quality.

A life tree showing the impact of Wallace Coulter’s inventions and discoveries up to the date of his death in 1998. On the right are some of the 85 patents issued to Wallace Coulter; technologies that made major contributions to the field are

shown on the left side of the tree. In the centre are some of the most important technologies with which Wallace Coulter was directly involved or, as in the case of monoclonal antibodies, in which he immediately saw the opportunity and

built significant businesses and technologies that capitalized on these inventions

Robinson, J. Paul. "Wallace H. Coulter: decades of invention and discovery." Cytometry Part A 83.5 (2013): 424-438.

coulter counter

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