1. Coulter counter Flashcards
What is a Coulter counter?
a device that counts the corpuscular elements of blood in a given volume electronically.
How does Coulter counter work?
A blood sample, diluted with physiological saline solution (electrolyte, good conductor) is pumped through capillary (a narrow aperture) at the bottom of measuring tube
-> while constant electric current intensity is maintained between the two parts of the dispersion.
If a particle enters the aperture, the effective cross-section is narrowed
→ resistance increases and a voltage pulse (spike) proportional to the volume of the particle is generated.
What is the Principle of operation of the Coulter counter.
- A well- conducting dilute dispersion of non-conducting particles is pumped through a small aperture (capillary), while constant electric current intensity is maintained between the two parts of the dispersion.
- If a particle enters the aperture, the effective cross-section is narrowed, hence resistance increases and a voltage pulse (spike) proportional to the volume of the particle is generated. (U=RI)
Principle of operation of the Coulter counter.
A blood sample, diluted with physiological saline solution (electrolyte, good conductor) is pumped into the measuring tube through a narrow aperture (capillary) at the bottom of the measuring tube.
If the electrolyte level reaches the measuring electrode inside the measuring tube, what will happen to measuring circuit and its resistance?
- The measuring circuit becomes closed
- Its resistance is given by the resistance of the electrolyte in the capillary (since all other resistances are negligible)
Principle of operation of the Coulter counter.
A blood sample, diluted with physiological saline solution (electrolyte, good conductor) is pumped into the measuring tube through a narrow aperture (capillary) at the bottom of the measuring tube.
As long as pure electrolyte fills the capillary, the constant measuring current intensity generates ___ (4w) on its resistance.
a constant voltage drop
Principle of operation of the Coulter counter.
What happen to the conductive cross-sectional area if a particle of different resistivity (usually a bad conductor, p≈ ∞) enters the capillary?
the conductive cross-sectional area is suddenly decreased (see diagram, bottom right).
Principle of operation of the Coulter counter.
If, however, a particle of different resistivity (usually a bad conductor, p ≈ ∞) enters the capillary, then the conductive cross-sectional area is suddenly decreased (see diagram, bottom right).
What is the consequence? Why does it happen?
a momentary increase of capillary resistance (R‘capill >Rcapill)
Because the measuring current is constant (Igen), according to Ohm’s law (U ‘ = R’capill·Igen) the voltage is increased during the passage of the particle through the capillary.
Principle of operation of the Coulter counter.
Because the measuring current is constant (Igen), according to Ohm’s law (U ‘ = R’capill·Igen) the voltage is increased during the passage of the particle through the capillary. Thus, each passage of a particle induces ____ (3w)
a voltage pulse.
Principle of operation of the Coulter counter.
What is the relationship between the amplitude of pulse and volume of particle?
The amplitude of the pulse is proportional to the volume of the particle.
Principle of operation of the Coulter counter.
The process of pumping up the solution continues until the electrolyte level reaches ____ (3w)
The auxillary electrode
Principle of operation of the Coulter counter.
The process of pumping up the solution continues until the electrolyte level reaches the auxiliary electrode (diagram), and its circuit switches to the ___ position.
pumping out
Principle of operation of the Coulter counter.
During the emptying of the measuring tube (the device is still counting), the electrolyte ___ the measuring electrode
leaves
Principle of operation of the Coulter counter.
If the measuring circuit breaks, the counting __
stops
Principle of operation of the Coulter counter.
In the end, what is the volume determined by?
- The heights of the measuring and auxiliary electrodes
- The cross-section area of the measuring tube is counted twice (Vup+Vout)
In the Coulter counter that we use in our measurements (PICOSCALE), the amplitude of the pulses is enhanced with an ___.
amplifier
In the Coulter counter, when red or white blood cells are measured, what does discriminator do?
when red or white blood cells are measured, the discriminator level (Ud ) is adjusted so that the circuit filters the pulses of the platelets as well (Fig. 4).
Coulter counter
What is the relationship between the number of pulses and the concentration of corpuscular elements?
They are proportional to each other
Coulter counter
What does the proportionality constant depend on?
It depends on the dilution of the blood sample, the measuring volume, and the factor of the scaling circuit
Coulter counter
Carefully filtered physiological saline solution, used for the dilution of the blood sample maintains (1)____(4w), and provides the (2)____ necessary for the measurement.
- The corpuscular elements intact
- conductivity
Coulter counter
The recommended dilution ratio for red-blood-cell counting is 1: 63 000. However, the measuring volumes of the instruments (see Fig. 2) may differ slightly, thus the measured concentrations may differ from the true value.
For this reason, the manufacturer always provides a calibration suspension called ___ for verification and calibration of the operation of hematology automated analyzers.
redcal
Coulter counter
The pulse-amplitude analyzer used in the hematology automated counter is based on the principle of the ___
differential discriminator (DD)
Coulter counter
The pulse-amplitude analyzer used in the hematology automated counter is based on the principle of the differential discriminator
In the DD circuit instead of one discrimination level (as in the ID), there are ___ levels.
2
Coulter counter
What does the differential discriminator (DD) do?
It allows to count only those signal pulses, for which the amplitude of the pulse falls between the two DD levels, into the so-called channel (it does not take into account those pulses that are either below the lower or above the higher discrimination level of the channel)
Coulter counter
differential discriminator (DD)
→ The position of the channel (1___, Ud) and its width are (2)___.
→ Size distribution of the particles can be mapped by a series of measurements with changed (3)___ of the channel, and it can be shown in a (4)___.
- central discrimination level
- adjustable
- position
- histogram
Coulter counter
What is the multichannel analyzer?
- The device that operate many DD-circuits simultaneously next to each other with neighboring channels
- Analyzer of the instrument called LABORSCALE together with the 64 channel pulse- amplitude analyzer
- enables us to display the size distribution of the corpuscular elements of blood
Coulter counter
Plan of measurement
- measure the size distribution of the corpuscular elements of the redcal suspension by using LABORSCALE
- The frequency distribution (histogram) will appear on the cathode-ray tube display of the analyzer.
- A blood sample will be analyzed with the automated hematology analyzer (based on principal of Coulter counter)
Coulter counter
A blood sample will be analyzed with the automated hematology analyzer.
Amplitude analysis of the pulses makes the determination of many other parameters possible.
How does automated hematology analyzer work when it comes to the counting of RBCs?
the discriminator level is set so that the platelets, which have a smaller size, are not counted
(The average size of the white blood cells is slightly greater than that of the red blood cells, but it does not cause significant error,)
Coulter counter
A blood sample will be analyzed with the automated hematology analyzer.
Amplitude analysis of the pulses makes the determination of many other parameters possible.
How does automated hematology analyzer work when it comes to the counting of RBCs?
The red blood cells need to be hemolyzed, as their size ranges overlap and because the large number of the red blood cells.
Coulter counter
A blood sample will be analyzed with the automated hematology analyzer.
Amplitude analysis of the pulses makes the determination of many other parameters possible.
How does automated hematology analyzer work when it comes to the counting of Platelets (thrombocytes)?
Platelets (thrombocytes) are much smaller than other, larger corpuscular elements, thus there is no need for other separation procedure to count them.
Coulter counter
For the equipment used in this practice (Sysmex F-800), 20 µl of blood needs to be diluted in two steps: first, at a ratio of 1: 500, then a further 100 times (to the final 1: 50 000 dilution ratio).
What are the 2 dilution?
- White-blood-cell counting is done from the first dilution (a hemolyzing agent is added), and hemoglobin concentration is determined photometrically.
- Red blood cells and platelets are counted from the second dilution.
(The two dilutions are measured with two separate detectors (measuring capillary and electrodes).)
What is The calibration value?
the ratio of the actual to the measured particle concentration.
Plan of measurement
Measurements on the sample of redcal suspension of known concentration cactual = 4.61·106/l (Fig. 7, BLACK, x10 button at OUT position)
- actual concentration is the redcal concentration
- Measure the number of counts (n) of the sample with known concentration
- Determine the calibration value (h)
4.
Plan of measurement
Measurements on the sample of redcal suspension of known concentration cactual = 4.61·106/l (Fig. 7, BLACK, x10 button at OUT position)
Amplitude-frequency spectrum
Plot the graph number of pulses n versus ID level Ud
→ Calculate the pulse frequencies for amplitude classes
→ Draw the histogram of the data calculated in step (f ) (∆n pulse number versus Ud ID level, Fig. 8, lower graph).
