Patent Application
20110027825
The present invention relates to a method for determining size distribution of cells in a sample. The sample preferably comprises a mixture of liquid and cells. The sample may be a blood sample.
When examining a sample, e.g. a blood sample, there is a need for a high resolution method of counting and size determining cells in such samples.
One way of counting or characterizing cells is by using a particle characterisation apparatus in which particles suspended in a liquid are passed through an orifice, in principle one by one, to enable the characterisation of the particles, for instance by Coulter counting.
It is well-known that particles travelling through a small orifice can be characterised with respect to size, concentration and conductivity by the use of an electrical impedance technique, widely known as the Coulter sizing (see V. Kachel, “Electrical Resistance Pulse Sizing: Coulter Sizing”, Flow Cytometry and Sorting, Second Edition, pp. 45-80, 1990 Wiley-Liss).
Counting and sizing of particles by the Coulter principle is an internationally respected method that is being used in most haematology-analysers and particle counting equipment. The method is based on measurable changes in the electrical impedance produced by non-conductive particles in an electrolyte. A small opening, called the “aperture” or “orifice”, connects two electrically isolated chambers, where electrodes have been provided to contact the electrolyte. The orifice applies a restriction to the electrical path, whereby a sensing zone is established through which the particles are aspirated. In the sensing zone each particle will give rise to a displacement of the surrounding electrolyte, thus blocking part of the current-path and giving rise to a voltage pulse. By this method several thousand particles per second can be characterised with high precision.
It is also well-known that the peak amplitude of the voltage pulses generated by the particles are closely correlated to the size of the particles, and therefore it is desirable to be able to determine the peak amplitude of voltage pulses in a simple and reliable way and at a low cost.
However, if a high resolution is desired, the traditional way of achieving a higher resolution is to implement hardware with more channels for measuring and categorising pulses, resulting in an increase in hardware cost and complexity. A new method for determining pulse height distribution lowering the cost and providing more simple hardware is required.
The present invention relates to a method of determining pulse height distribution by using an apparatus comprising: an analogue to digital pulses height categorisation unit comparing the pulse to analogue threshold voltages and counting each event within each pulse height category using a micro controller, the method comprising the steps of:
The selected threshold voltages are applied to the analogue to digital pulses height categorisation unit before performing a measurement. The threshold voltages may also be threshold currents or simply threshold values.
Surprisingly is has been found that performing methods for counting cells in accordance with the method defined above using different or shifted or moved threshold voltages yield a greatly improved precision.
The first set of threshold voltages may be chosen from a look-up table, be entered by a user, calculated or determined on the basis of a known particle size distribution, determined using other means or any combination of the above.
The result of a measurement may be recorded in a memory unit in or electrically connected to the apparatus used for performing the method according to the present invention. The memory unit may be of a temporary sort, such as a buffer or the like.
Also, the recorded data from the measurement may be stored in or transferred to other data storage devices, such as hard drives, optical drives etc.
A new set of threshold voltages are chosen for step iii. The new set of threshold voltages includes at least one new threshold voltage, i.e. the at least one new threshold voltage is different from any of the threshold voltages from the first set. The new measurement may be performed in substantially the same way as the first measurement.
The determination of the cell size distribution is based on the first measurement and the new measurement. The determination may for instance be performed using a back-substitution which is preferably a numerical procedure performed on the set of measurements, in the above case two measurements, but generally the back-substitution may be performed on the entire set of measurements. Generally speaking the determination may be a reverse calculation performed by means of an adapted algorithm reconstructing the original cell distribution on the basis of the set of measurements.
The apparatus mentioned above may have components for obtaining the pulse heights implemented using an integrated circuit, a field programmable gate array or an application specific integrated circuit, or a combination thereof.
In one embodiment of the present invention the, pulse height determination unit may comprise a first plurality of comparators with a common input for analogue to digital conversion of the electronic pulses, a first plurality of latches wherein the inputs of the latches are connected to the outputs of respective comparators for recording passage of the corresponding threshold voltages by the rising edge of a pulse, a priority encoder connected to the latch outputs for determination of a pulse height category consisting of pulses with a pulse height within a pulse height interval defined by respective threshold voltages, and a micro controller that is adapted to count the number of pulses within each pulse height category.
It is an advantage of the present invention that the threshold voltages may be individually adjusted as desired. For example, it is not required that the threshold voltages are equidistant. If the possible size distribution of the particles is known, it is possible to select a number of threshold voltages that are adjusted for optimum determination or detection of the actual size distribution of the particles. For example, in analysis of whole blood, it is desirable to count the number of three types of blood cells erythrocytes, leukocytes and thrombocytes. Their size, expressed as equivalent diameter or volume, ranges from app: 1.2 μm or 1 fl (1 fl=10-15 l) for the smallest thrombocytes to app. 9 μm or 400 fl for the largest leukocytes.
Information on the content of leukocytes, their subpopulations and thrombocytes is an important tool for the physician in order to diagnose different diseases and monitor treatment. Furthermore, the concentration of haemoglobin, directly related to the number of erythrocytes, in the blood sample is also of great importance.
Thus, the number of erythrocytes, leukocytes and thrombocytes may be counted utilising the pulse height analyser as described above with threshold voltages that are selected and adjusted in accordance with the known sizes of the erythrocytes, leukocytes and thrombocytes, e.g. by positioning threshold voltages in between corresponding mean values of the individual particle size distributions.
The first set of threshold voltages may define a first threshold voltage span and the new threshold voltages may define a new threshold voltage span. The first threshold voltage span and the new threshold voltage span may overlap or alternatively the first threshold voltage span and the new threshold voltage span do not overlap or further alternatively the first threshold voltage span and the new threshold voltage span have one common point. The first threshold voltage span and the new threshold voltage span may further have more than one common point. The span is preferably defined by the highest threshold voltage and the lowest threshold voltage in a given set.
The back-substitution mentioned above may be chosen dependent on the interrelation of the threshold voltages of the individual sets of threshold voltages.
The number of threshold voltages in a threshold voltage set may depend on the number of elements in the first plurality mentioned above. The number of threshold values may be 2 to 20, such as 5 to 15, such as 8 to 12, such as 2 to 5, such as 5 to 8, such as 8 to 10, such as 10 to 12, such as 12 to 15, such as 15 to 18, such as 18 to 20, such as 8. The actual number of threshold values may depend on the apparatus used for performing the method according to the present invention. It is an advantage of the present invention that the method may be implemented as a software program executed on either existing hardware or in the alternative on especially developed hardware.
It is an advantage of the present invention that the steps iii) and iv) may further be performed 1 to 20 times such as 5 to 15 times such as 8 to 12 times such as 2 to 5 times such as 5 to 8 times such as 8 to 10 times such as 10 to 12 times such as 12 to 15 times such as 15 to 18 times such as 18 to 20 times such as 10 times. The number of times that the measurements are repeated may depend on desired accuracy and/or amount of test fluid. As the test fluid is to be passed through an orifice there may be a limited amount of fluid available, alternatively the fluid may be re-circulated. It may be further advantageous that the fluid comprises a substantially homogenous distribution of particles, i.e. blood cells or the like.
In particular embodiments of the present invention the new set of threshold voltages in each repetition may be different from any previously chosen set of threshold voltages. A set of threshold voltages may considered different from any other set when at least one threshold voltage is different from any other previous threshold voltage in the set of threshold voltages. Alternatively all threshold voltages of each set may be different from any other threshold voltages of any other set of threshold voltages, i.e. the same threshold voltage is not reused in any set of a given set of measurements.
It is particularly advantageous that a new set of threshold voltages may be calculated using the equation:
T
i,j+1=αi,j+1Ti,j+βi,j+1
where:
each set of threshold voltages include N number of threshold voltages,
Ti is the i'th threshold value, i=0 to N−1
j is the j'th threshold voltage set, j=1 to the number of repetitions of steps iii) and iv).
In some embodiments of the present invention the equation may be modified to:
T
i,j+1=αi,j+1Tx,j+βi,j+1
Where Tx,j are the threshold voltages of the x'th set. In some embodiments the threshold values may be calculated based on the first set of threshold voltages, i.e. x=1 in the above equation. In other embodiments the threshold values may be calculated on the previous set of threshold values.
The threshold values may be pre-calculated using any of the above equations and subsequently stored in a look-up table, in a database or any other suitable storage. Alternatively a set of threshold voltages may be calculated during or shortly prior to each repetition of the measurement steps above, i.e. calculated on-the-fly or during the measurement. Further alternatively the threshold voltages may be inputted by a user and stored for use when performing the measurements.
Depending on the distribution of the actual sizes of the cells to be counted, the first threshold values may be equidistantly distributed or the first threshold values may be distributed at non-equidistant distances. The subsequent threshold values do not need to have the same characteristics as the first threshold values. As an example the equation above may result in a series of threshold voltages where a first set of threshold values are equidistantly distributed, whereas the second set, calculated on the basis of the first set, is not equidistantly distributed.
The following sets of threshold values, as stated above, may be calculated using any of the above mentioned equations. In the equations the α's may have any positive real value or be zero, and the β's may have any real value, i.e. negative, positive or zero. In an embodiment where all α values are 1 or 0, the value β will cause the span of the threshold values to be shifted either up of down depending on the sign of β.
In most embodiments the first set of threshold voltages start at some distance from zero, as the size of the particles to be characterised influence the voltages created, and these voltages are usually different from zero.
As mentioned above the method according to the present invention may be implemented as a software program, and the present invention thus further relate to an apparatus comprising a computer software implementation of the method according to the present invention. Also the present invention relates to a data-carrying medium comprising a computer software implementation of the method according to the present invention. The data carrying medium may be a hard drive, a flash drive, an optical storage disk, such as a compact disk (a CD) or a digital versatile disk (a DVD) or any other suitable data carrying medium.
In the following the invention will be further described and illustrated with reference to the accompanying drawings in which:
For the measurement a very high resolution was applied. The test equipment featured 800 discrete channels. The known method of counting cell populations is a high cost method requiring complicated hardware and software.
The x-axis in
The two cell populations under investigation in
Due to the lower resolution it is difficult to determine the exact split, at 18, between the two populations.
A system having 8 channels, as was also the case with the measurement in
The system or apparatus comprises 8 comparators with a common input for analogue to digital conversion of electronic pulses, 8 latches wherein the inputs of the latches are connected to the outputs of respective comparators for recording passage of the corresponding threshold voltages or values by the rising edge of a pulse, a priority encoder connected to the latch outputs for determination of a pulse height category consisting of pulses with a pulse height within a pulse height interval defined by respective threshold voltages, and a micro controller that is adapted to count the number of pulses within each pulse height category.
The x-axis of the chart in
The method is performed by first selecting a first set of threshold voltages followed by performing a first measurement using the first set of threshold voltages. Then selecting a new set of threshold voltages different from the first set of threshold voltages, and performing a new measurement using the new set of threshold voltages.
The measurement is repeated 5 times, illustrated by the 5 bars 26, 28, 30, 32 and 34. In each repetition of the measurement the threshold voltages are moved or shifted according to the general equation:
T
i,j+1=αi,j+1Ti,j+βi,j+1
where each set of threshold voltages include N number of threshold voltages, Ti is the i'th threshold value, i=0 to N−1, j is the j'th threshold voltage set, j=1 to the number of repetitions. In the example in
Each slot of the x-axis represents a channel. Each channel comprises bars comparative to the number of repetitions of the measurement, here 5 repetitions are performed. In this example the threshold values are shifted or moved an equidistant distance.
Repeating the measurement with different threshold values, calculated using the equation above, gives a surprisingly significantly improved result compared to the method described in relation to
The method according to the present invention is relatively easy to scale depending on the desired resolution. When using the known method, as described in relation to
Comparing the result in
A curve 33 illustrates the particle size distribution calculated on the basis of the result illustrated by the bars in
Before the bar chart of
In all examples given in
As described above in stead of using more fixed thresholds for the size classification, the adjustment of the current or voltage thresholds may be used for stepping the thresholds during the testing or measuring e.g. with a fixed time sequence. The change in each box will reflect the resolution of a size classification with a resolution matching the step. It is thus the size of the step that determines the resolution.
One simple example is the case where there is only one classification defined by a single threshold. With a given constant and limited distribution of pulses heights, such as illustrated by
P(i)=C(i)−C(i+1), i=1 to N−1
The result is shown in
In an embodiment where 8 classes are used to categorize the pulse heights, it may be advantageous to use equidistant thresholds. In this way the steps may be chosen to match the width of the classifications divided by the number of steps used.
Furthermore, it may be advantageous to match the thresholds such that the full pulse height distribution is always included. This way the total count of the pulses in each time frame should remain the same, which is easily verified. Given a pulse height distribution as illustrated in
P(i+5*(j−1))=C(j:i)/5, j=1 to 8, i=1 to 5
The result of the calculation on the basis of the measurement is shown in
In some embodiments non-equidistant thresholds may be used. The back substitution may be more complicated and depends on the variation of the distances. The easiest way to overcome this problem is to resolve each size classification into classifications of the same threshold distance as the shortest distance, this should preferably be an integer number. The summed content of the partitioned classifications are equal to the content of the original size classification but may be distributed unequally by interpolation with the neighbour size classifications.
Further, during a counting process counting may vary during the time of measurement. For instance the counting may decrease or increase slightly as flow through the aperture changes. In such a situation it may be advantageous to use a repeated sweep method. The method may comprise that the step time is reduced to ⅕ or 1/10 of the total counting time. After the last step of a sweep is complete the whole procedure is started over in a new sweep until the full counting time is reached. Thereby a series of sweeps are performed and the slow change in counting is distributed to all of the counting steps.
In one example the pulse height distribution in an 8-class resolution counting with 5 steps and 10 sweeps could this be noted as C(j:i:k), where j denotes the j'th class, i denotes the l'th step and k denotes the k'th sweep. The 40-class distribution can be found by calculating class content, P(i) as:
If the counting is based on flow, the total time used for counting cells in a specific volume may vary slightly. If in the above example a sweep has not come to an end when counting is stopped holes in the data representing the cell size distribution may occur. It is contemplated that this may be corrected or compensated for by not using the last, incomplete sweep, but instead make use of a correction of the contents of the preceding sweeps to match the total counting. If the total counting of all size classes including the last incomplete sweep is denoted Ptot, the correction may be expressed as:
where j=1 to 8, i=1 to 5 and last is the number of the last sweep encountered when the counting was stopped.
In an advantageous embodiment a series of sweeps may be performed within a first time interval. This may for instance be a period of XX minutes. The flow rate may vary over the first time interval, i.e. the flow rate may change slightly during the measurements. The method may further comprise reducing for each sweep in the series of sweeps the time used for the steps in one sweep. This means that the time spent for one sweep is longer than the time spent on the following sweep and/or sweeps.
Advantageously the time used for one sweep is reduced by a factor in the interval 1/20 to ¼ for each sweep. The time spent for sweeping may be reduced by a specific factor for each sweep. As an example a first sweep is performed in 10 seconds, and the immediately following sweep is performed in 8 second, thereby reducing the sweep time by ⅕.
In another advantageous embodiment a series of measurements may be performed using a first volume, i.e. the measurement is continued until a specific volume has been investigated. The flow rate may vary over the first time interval, meaning that the total time spent may vary and the number of sweeps may not constitute a complete number of sweeps. The method may further comprise correcting the last sweep using the formula:
where Ptot is the incomplete sweep, j is the number of classes, i is the step number, max(i) is the number of steps, k is the number of sweeps and last is the number of the last sweep performed.
| Number | Date | Country | Kind |
|---|---|---|---|
| PA200800315 | Mar 2008 | DK | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/DK2009/000057 | 3/2/2009 | WO | 00 | 10/22/2010 |
