Patent Document

This application is a Divisional of U.S. application Ser. No. 08/754,823 filed Nov. 21, 1996 still pending. 
    
    
     FIELD OF THE INVENTION 
     The invention relates to particle or cell sizing and counting apparatus and to methods of operation thereof. In particular, the invention relates to apparatus which uses a technique of measuring the impedance at an orifice to determine the volume of a particle passing through the orifice. 
     BACKGROUND OF THE INVENTION 
     It is known from EP 0162607 to determine the size of a particle from the variation in impedance between a pair of electrodes in an electrolyte due to particle flow through an orifice in a flow restrictor disposed between the electrodes. An inherent problem of this type of system however, is that partial or complete blockage of the orifice can occur during measurements which requires that the flow restrictor comprising the orifice must be removed in order to be cleaned to allow further measurements. Additionally, if only partial blockage occurs the observed distribution of particle sizes in a sample will be affected due to prevention of flow by the blockage of larger particles through the orifice. This problem is particularly prevalent if one wishes to use a small orifice diameter of say five times the average particle size to enable good accuracy of particle sizing results. 
     SUMMARY OF THE INVENTION 
     The invention seeks to avoid or at least mitigate problems of the prior art including providing apparatus which detects blockage and or deblocks the orifice especially in the event of partial or complete blockage. 
     According to first aspect of the invention there is provided apparatus for determining the size of a particle or cell within a fluid, comprising a sample chamber for the fluid, a flow restrictor comprising an orifice, a pair of electrodes disposed on opposite sides of the orifice and means for measuring a signal representative of the impedance variation between the electrodes thereby to determine the size of a particle within the fluid passing through the orifice, and further comprising means for detecting blockage of the orifice, whether partial or complete. Beneficially the detection of a partial or complete blockage alerts the user to data corruption. 
     Preferably, the detecting means comprises means for monitoring the signal, which means detects occurrence of a predetermined variation of the signal indicative of blockage of the orifice. For example, particle passage through the orifice causes a signal pulse which is measured by the measuring means and wherein the monitoring means determines a width of the signal pulse and compares this width with a predetermined value thereby to detect partial or complete blockage of the orifice. The predetermined pulse width value can be determined from an average of previously measured pulse widths. 
     In another form, the monitoring means monitors the mean base line value of the signal to determine if a significant drift in mean base line value occurs which is indicative of partial or complete blockage of the orifice. 
     Alternatively or as well, the monitoring means compares the height of an individual signal pulse with a known value corresponding to a particle size in the order of or greater than a predetermined size such as the diameter of the orifice. Also, the monitoring means can comprise a saturation pulse or square wave detector. The square wave detector can compare the time for a pulse signal to pass through a first and second predetermined value and then return back through the predetermined first and second values. The square wave detector can be arranged to detect a characteristic recovery curve indicative of saturation of the measuring means. 
     In another form, the monitoring means measures the rate of occurrence of signal pulses and compares this rate with a predetermined rate. Also, the monitoring means can compare the number of detected signal pulses in a given time interval with a predetermined value, such as an average of previous measurements. 
     Beneficially, the monitoring means can analyse the background noise of the signal for predetermined variation, such as amplitude variation within a frequency range. 
     Further, the detecting means can comprise an orifice current detector for determining electrical current flow between the electrodes thereby enabling the monitoring means to compare orifice current with predetermined values. The detecting means can compare the orifice current value before or after measuring a signal or the mean of the two with an initial value measured before the signal measurement. The detecting means can determine if the difference is greater than a 10% increase or a 5% decrease, or if the difference is greater than 20% say. 
     Preferably, the measuring means comprises a 14 bit detector, or an analogue to digital converter having 14 bit resolution. Also, the apparatus preferably comprises means for applying a calibration signal to one of the electrodes and the monitoring means the signal across both electrodes, the monitoring means being adapted to compare the detected calibration signal with a pre-determined signal. The calibration signal can comprise a series of pulses. The calibration signal can be repeated a predetermined number of times to attempt to obtain an acceptable result with the predetermined signal, before providing an alarm to the user. Also, means for deblokcling the orifice by removing a particle at least momentarily held within the orifice can be provided. 
     According to another aspect of the invention there is provided apparatus for determining the size of a particle or cell within a fluid, comprising a sample chamber for the fluid, a flow restrictor comprising an orifice, a pair of electrodes disposed on opposite sides of the orifice and means for measuring a signal representative of the impedance variation between the electrodes thereby to determine the size of a particle within the fluid passing through the orifice, and further comprising means for deblocking the orifice by causing movement of a particle held at least momentarily within the orifice. Beneficially the deblocking means can effect in situ removal of the blockage, therefore, the apparatus allows continuous sampling without requiring the user to dismantle the sample chamber or remove the flow restrictor for cleaning when a blockage occurs. 
     Preferably, the deblocking means operably creates ultrasonic vibrations in the fluid in the region of the orifice. A series of electrical pulses can be applied to one of the pair of electrodes. The pulse frequency is preferably greater than 15 kHz, and preferably up to about 20 kHz. Beneficially, the flow restrictor can comprise material that exhibit the piezo-electric effect, thus to enhance the deblocking effect in the presence of electrical pulses. 
     Also, the deblocking means can comprise fluid propulsion means for effecting fluid flow at the flow restrictor. Preferably, the fluid propulsion means directs fluid substantially in the reverse direction to fluid flow during signal measurements. 
     Beneficially, means within the chamber can be used for directing fluid from the fluid propulsion means towards the flow restrictor. For example, a tapered region of the chamber can be used. 
     Another aspect of the invention provides a sample chamber housing for particle sizing apparatus comprising a pair of recesses each adapted to receive an electrode, an electrode in each recess, and a two-part sealant and adhesive for the electrodes. The adhesive can comprise a structural acrylic adhesive. The sealant can comprise a silicone based sealant such as RTV silicon rubber. The recess is preferably defined in a body of a polymer such as acrylic. 
     A further aspect of the invention provides a flow restrictor for particle sizing apparatus, comprising an orifice allowing flow of particles therethrough which orifice is tapered. The taper is preferably in the order of 5 to 30% of the mean orifice diameter. 
     A further aspect of the invention provides a flow restrictor wherein the material defining the orifice is flexible. 
     A yet further aspect of the invention provides use of a 14 bit analogue to digital converter in particle sizing apparatus. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     Embodiments of the invention will now be described by way of example only, with reference to the accompanying drawings in which; 
     FIG. 1 is a schematic perspective view of a sample wand and a sample chamber housing forming part of the apparatus according to the invention; 
     FIG. 2 is a schematic front elevation view of the sample wand alignment guide as shown in FIG. 1; 
     FIG. 3 is a schematic block diagram of the fluid control apparatus according to the invention; 
     FIG. 4 is a sectional view through a sample chamber according to the invention; 
     FIG. 5 is an end view from inside the apparatus of the sample chamber shown in FIG. 4; 
     FIG. 6 is a partial sectional view of the sample chamber shown in FIGS. 4 and 5; 
     FIG. 7 is a schematic perspective view of an orifice in a flow restrictor; 
     FIG. 8 is a schematic perspective view of a second flow restrictor according to the invention; 
     FIGS. 9 a, b  and  c  are is a schematic side views of the restrictor of FIG. 8 in three different modes of use. 
     FIG. 10 is a schematic block diagram of part of the electronic control system for the apparatus according to the invention; 
     FIGS. 11A and 11B comprise a circuit diagram for part of the amplification circuit for the apparatus according to the invention; and 
     FIG. 12 is a schematic view of a signal pulse detected and analysed by the apparatus according to the invention. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Referring to FIG. 1 there is shown part of a particle sizing apparatus  10  according to the invention comprising a main housing  12  from which protrudes a sample chamber housing  14  having an inlet  16  through which a sample passes into first chamber  52  shown in FIG.  4 . Additionally, a wand guide  18  protrudes from housing  12 . Wand guide  18  comprises a vertical groove or channel  20  which enables alignment of nozzle  24  of hand-held wand  26  with inlet  16 . Hand-held wand  26  further comprises a replaceable tip  28 , a button  30 , fluid inlet pipe  32  and electrical connection  34 . As shown in FIG. 2 wand guide  18  comprises a pair of detectors  22  such as optical detectors to determine if wand nozzle  24  is in place within groove  20 . 
     Referring to FIG. 3 there is shown a schematic drawing of the fluid control system  36  used in apparatus  10 . In fluid control system  36  a diluent reservoir  38 , which can house say three liters of electrolyte for example, is connected along one path to a three-way valve  40  which is connected at one outlet to a syringe drive  42  which can comprise a 12 volt stepper motor for example, and at another outlet is connectable to wand  26  by fluid inlet pipe  32 . 
     Reservoir  38  is also connected via a T-connector  44  to a diluent sensor  41 , which can be an optical device for determining if diluent is present. A pump  46  such as a 12 volt DC peristaltic pump, drives diluent to sample chamber housing  14  via a Y-connector  48 . 
     One outlet of Y-connector  48  is connected to tube  50  having a valve  51  such as an electrically operated pinch valve, which controls flow of diluent through tube  50  to a first chamber  52  forming part of sample chamber housing  14 . The other outlet from Y-connector  48  passes through tube  54  which has a valve  56 , again such as an electrically driven pinch valve, which controls flow of fluid through tube  54  into a second chamber  58  of the sample chamber housing  14 . First and second chambers  52  and  58  respectively, are separated by a flow restrictor  60  having an orifice  63  (see FIGS. 7,  8  and  9 ). Sample chamber  14  is shown in greater detail in FIGS. 4 to  6 . 
     System  36  further comprises a suction system comprising a pair of vacuum pumps  62 . 
     Each pump is connected to a vacuum reservoir  64  via a valve  66 , such as an electrically driven pinch valve. Purge tubes  68  pass almost to the bottom of reservoir  64  thereby to enable expulsion of any liquid in reservoir  64  via at least one of valves  66 , pump  62  and outlet  70  thus for disposal. 
     Reservoir  64  is further connected via tube  72  to the second chamber  58  of housing  14 . Tube  72  further comprises a valve  74  such as a pinch valve and a pressure transducer  76  such as a Honeywell Controls type 141PC05G device. Reservoir  64  is further connected to the first sample chamber  52  via tube  78  having a valve  80 . 
     Referring to FIGS. 4 to  6 , sample chamber housing  14  is shown which preferably comprises an outer shield  15  against electromagnetic radiation such as a metallic casing. Inlet  16  leads to first chamber  52  which is separated from second chamber  58  by flow restrictor  60 . The chambers can be made in a body of inert material such as an acrylic or other plastic. The flow restrictor can comprise a crystaline material such as ruby, sapphire, or aluminium or a polymer having a orifice  63 . As shown in FIG. 7 orifice  63  has length L and diameter D, for example a length of 80 microns and diameter of 30 microns is preferred for certain sizes or volumes of particle P which during measurement flow through orifice  63  along direction F. However, other sizes of orifice are possible. Diluent tube  50  enables diluent to pass into first chamber  52  whilst tube  78  enables evacuation of the first chamber. 
     Similarly tube  54  allows diluent to pass into second chamber  58  and tube  72  enables the evacuation thereof. In practice the tubes can comprise different types of passageways, channels and connectors as appropriate. Inlets to sample chamber housing  14  are shown in FIG.  5 . 
     In FIG. 6, it can be seen that a recess  61  can be provided for holding flow restrictor  60  thereby to separate chamber  52  and  58 . O-rings can also be used to seal the edges of flow restrictor  60 . Additionally, electrodes  82  and  84  are shown effectively on opposite sides of flow restrictor  60 , i.e. one in each of chambers  52  and  58 . The electrodes can be made of platinum for example and are connectable to an electronics control system via connector  86 . 
     It has been found particularly useful to attach electrodes  84  and  82  to housing  14  using a two-part sealant and adhesive. This is in order to prevent leakage of fluid from chambers  52  and  58  and maintain the electrode in a correct position in spite of attaching and detaching chamber housing  14  from main housing  12 . Preferably, the electrodes are mounted in a recess in housing  14  which can be made of acrylic or other polymer material. The upper part of the recess, that is adjacent the fluid chambers, is sealed with a water resistant and vacuum seal material such as a silicon based sealant, for example RTV silicon rubber. A second stage of adhesive is placed below the sealant to attach the electrode to the walls of the recess. A suitable adhesive is a structural acrylic adhesive. In a preferred form, chamber housing  14  is made from acrylic, the electrodes have an outer platinum coating or are made of platinum and the two-stage sealant and fixing comprises RTV silicon rubber and structural acrylic adhesive. 
     Preferably, chamber housing  14  is easily detachable from main housing  12 . Accordingly, electrical connector  86  is preferably a simple bayonet-type connector comprising male components adapted to fit into a female socket on housing  12  for example. Additionally, as shown in FIGS. 4 and 5, tubes  72 ,  54  and  78  allow for easy mounting on housing  12  and can for example be the female part of a male/female connection. Accordingly, chamber housing  14  can be attached and removed from main housing  12  in a simple push/pull operation. 
     Referring to FIGS. 8 and 9 there is shown a second embodiment of a flow restrictor  60  according to the invention wherein orifice  63  is tapered. For example, the aperture on one side of restrictor  60  could be 32 to 38 microns in diameter whilst the orifice on the other side could be 30 microns. Accordingly, a taper of say 2 to 8 microns in the diameter of the orifice is affected across its length of say 80 microns. Preferably, the narrower orifice faces the second chamber  58  such that in use, as shown in FIG. 9 a,  where the restrictor  60  is made of resilient material, it flexes creating an orifice  63  having approximately parallel sides thereby enabling laminar flow through the orifice. The rest position in shown in FIG. 9 b  and a blow-back or clearing operation position is shown in FIG. 9 c.  The clearing operation is described in more detail later. 
     Preferably, the flow restrictor is made of a polymer such as a non-fluoride polycarbonate such as PET or polystyrene. The orifice can be laser etched into the flow restrictor. 
     Preferably, to assist in cleaning a blocked orifice  63 , second chamber  58  comprises a tapered region  59  which narrows towards flow restrictor  60  (see FIG.  4 ). Additionally, preferably tube inlet  54  to chamber  58  is axially aligned with orifice  63  so that fluid passing into chamber  58  is directed to effect turbulence at flow restrictor  60  and possibly to cause reverse flow of diluent through to first chamber  52 . The tapering of chamber  58  assists in this reverse flow and additionally causes circulation of diluent in chamber  58  assisting in the cleaning thereof. Further, a flexible restrictor  60  provides the enhanced clearance configuration shown in FIG. 9 c.    
     Referring to FIG. 10 there is shown a schematic block diagram of an electronics control system  88  for the apparatus according to the invention. System  88  comprises an amplifier stage  92  for detecting and amplifying a signal from electrodes  82  and  84 . 
     A signal power supply unit  94  is provided which generates a plus or minus voltage to be applied across electrodes  82  and  84 , for example a voltage of 150 volts is preferred; across the electrodes however this can be somewhat less and can be about 30 volts. Amplifier board  92  provides an earth connection to shield  15  around sample chamber housing  14 , and is additionally connected to vacuum transducer  76  and a thermocouple or temperature probe  95  such as a National Semi-Conductor type LM35CAH device which can be located for measurement of the diluent temperature in second chamber  58 . 
     An amplified sample signal is communicated to a 3½ digit variable potentiometer  96  along line  98  from amplifier stage  92 . The potentiometer forms part of an attenuator board  100  which enables adjustment of the signal gain as necessary for the selected orifice size and application. The output from the attenuator board  100  is fed to a 14 bit analogue to digital converter on measurement board  102 . The electrode current, vacuum signal and temperature signal are fed along lines  104 ,  106  and  108  respectively from amplifier board  92  to an 8 bit analogue to digital converter on measurement board  102 . Measurement board  102  comprises a channel of 500 kHz or 1 MHZ conversions at 14 bits, local storage for a channel of 14 bit data, eight channels of 8 bit (slow) A-D conversion at 100 microsecond conversion, controlling status registers for these devices, 16 bits of digital output control and 16 bits of digital input control. 
     Lines  110  and  112  are provided between measurement board  102  and amplifier board  92  respectively to enable reversing of the voltage polarity across the electrodes  82  and  84  and also for commencing and stopping an electrode calibration sequence to be described later. 
     A series of digital input/output ports are provided from measurement board  102 . These are connected at lines  114  to  120  a stepper motor driver  43  which is in turn connected to syringe stepper motor  42 . Line  114  carries an opto signal from the syringe drive indicating that the syringe is in its home position. Clock, enable and direction signals are relayed to stepper driver  43  via lines  116 ,  118  and  120  respectively. 
     The other input/output ports on measurement board  102  communicate with driver board  122 . This board, for example, controls the opening and closing of valves  51 ,  56 ,  66 ,  74  and  80 . It also controls the diluent pump  46  and vacuum pumps  62 . The diluent sensor  41  and user wand button  30  are also connected to driver board  122 . 
     A power supply unit  90  provides requisite voltage to drive the system  88 . Also, a programmable device such as a computer  103  is provided to drive and communicate with boards  100  and  102  (or other elements as necessary). A peripheral device such as a display and/or printer  124  is also provided. 
     Referring to FIGS. 11 a  and  11   b  there is shown a circuit diagram for part of the amplifier board  92 . A particle signal amplification path comprises four low noise op-amps IC 10 , IC 11 , IC 12  and IC 13  having frequency selective components which allow bandpass filtration of the signal to remove DC and high frequency noise. Amplifier IC 11  has a gain adjustment potentiometer that can be set during manufacture and sealed. Amplifier IC 13  provides amplifier board  92  with a DC offset adjustment (say −3.2V) using the resistor and potentiometer network R 35 , R 36 , VR 3  and R 37 . An electrode ( 82  or  84 ) is connected at CN 1  and is AC coupled to op-amp IC 10  via capacitator C 38 . A signal generating voltage source  94  of say 150 volts in this example, connects to the signal path at PL 5  and is controlled by reed relay RL 1 . Resistors R 23  and R 22  form a high impedance path in comparison to the diluent electrolyte through the orifice making the source  94  appear as a constant current generator. 
     The amplified signal output at CN 2  passes along line  98  to attenuator board  100  shown in FIG.  10 . An orifice current signal can be derived from DC current flowing through the chain VR 4 , RL 1 , R 23 , R 22 , R 21 , R 47  and orifice  63  via CN 1 . The electrode voltage source  94  connects at PL 5  and a voltage proportional to the orifice current is expressed across VR 4  and R 47  and amplified by IC 14   a.  This is further conditioned at amplifier IC 14   b  and output to measurement board  102  via line  104  shown in FIG.  10 . 
     The operation of the system is as follows. The system is formally calibrated for a given electrode voltage, orifice diameter and diluent using inorganic machined particles of known size or volume. After calibration at manufacture, a further calibration verification can be conducted during use of the system as described later. Both first and second chambers  52  and  58  of the sample chamber  14  are washed by alternately opening valve  51  and  56 , and valves  80  and  74  respectively, and pumping diluent using pump  46 . For example, chambers  52  and  58  can be filled and emptied using the vacuum system including pump  62  and tube  72  and  78 , for example a series of three times before again filling chamber  62  and  58  to enable an electrical calibration test. A series of calibration pulses is applied to one of the electrodes  82  or  84  at CN 1  using  1 C 9  shown in FIG. 11 such as a CMOS  555  timer device. The output voltage at CN 1  can be controlled for example at 12 volt peak to peak thereby to enable an impedance measurement across the orifice which signal might for example be in the order of 1 millivolt for a nominal 30 micron orifice. The pulse width can be in the order of 20 to 40 microseconds for frequencies in the order of 15 to 20 kHz. The mean pulse height and number of pulses are monitored as part of the calibration verification, a total of three repeated calibration measurements can be attempted before the sequence is aborted and an error condition signal provided to the user for example at a VDU display or similar output device  124  connected to board  102 . The fluid content of the diluent reservoir  38  is then checked for example using a sensor  41  connected to board  122  shown in FIG.  10 . 
     Syringe  42  is then loaded by directing the three way valve  40  to relay fluid from reservoir  38  into syringe  42  and driving stepper motor  43  a calibrated number of steps in order to load the syringe unit with a known volume of diluent such as 1 milliliter. The user is then requested to place a disposable sterilised tip  28  on the end of nozzle  24  of wand  26  and press the button  30  to confirm. The user then places the disposable tip in the sample fluid and again operates button  30  and syringe  42  is driven down a pre-programmed amount thereby to draw sample fluid into the disposable tip  28 . 
     The user places the sample wand so that nozzle  24  is aligned in groove  20  and tip  28  protrudes into first chamber  52 . First chamber  52  is emptied by opening valve  80  and using pump  62 . The operator presses button  30  to initiate a dispense sequence wherein syringe  42  is driven with valve  40  directing fluid through pipe  32  such that the sample and diluent are emitted from wand  26  into first chamber  52 . If during this operation, detectors  22  determine that nozzle  24  is not in position or has moved away from this position in channel  20 , the system waits for the nozzle to be correctly re-positioned. If the sample aspiration, dilution and presentation to chamber  52  is not completed in a predetermined interval, eg 2 minutes, the measurement is aborted and the sample chamber cleaned and the process re-initiated. 
     It will be appreciated that it is possible by determining the extent of movement of syringe  42  in any direction to quantify, using stepper motor  43 , the amount of sample drawn up through tip  28  and the amount of diluent drawn in to syringe  42  to be mixed therewith, and hence the concentration of sample and diluent can be accurately determined. 
     Second chamber  58  is primed with fresh diluent through tube  54  by opening valve  56  and operating pump  46  to ensure that it is substantially full. The amount of diluent should be sufficient to enable an electrically conductive path between electrodes  82  and  84  across the flow restrictor  60 . Preferably an initial priming of second chamber  58  is conducted by passing diluent through tube  54 , as previously described, causing turbulence in chamber  58 . This acts to reduce the possibility of blockage of orifice  63 . 
     The vacuum reservoir  64  is evacuated of fluid using pumps  62  such that when valve  80  and  74  close a vacuum in the order of 130 mm Hg is established in reservoir  64  as read by transducer  76 . For example, one of pumps  62  can be turned off or the associated valve  66  closed, on reaching 90% of the predetermined vacuum pressure and using only one of pump  62  to achieve the predetermined level of vacuum. 
     Valve  74  is opened with valve  56  closed and accordingly some sample and diluent is drawn through the orifice  63  of flow restrictor  61 , that is between chambers  52  and  58 . The known voltage is applied to the electrode  82  (or  84 ). After a stabilising delay of say 2 seconds, the current through the orifice, i.e. between electrodes  82  and  84  is checked at line  104  (see FIG.  10 ). The current start value or initial value is stored for use in later comparative checks. 
     Preferably the large DC voltage is removed and a calibrator reference signal applied to electrode  82  (or  84 ) as previously described. 
     The known DC voltage, is applied to electrode  82  and the resulting signal amplified between CN 1  and CN 2  of FIG.  11  and output from the amplifier board  92  at line  98  is shown in FIG.  8 . An example of the type of pulse observed when a particle passes through orifice  63 , is shown in FIG.  12 . The pulse signal S has a height, or voltage, determined by the volume of the particle. The width W of the pulse is determined by the time for the particle to flow through the orifice, i.e. the flow rate and axial length L of the orifice. The signal also has a characteristic background noise N. 
     In one example of the operation of the system, the programmable device  103  sends a signal to measurement board  102  to initiate 14 bit data conversion at 16384 conversions every two microseconds each. The gain on the attenuator board  100  can then be adjusted as necessary to bring the signal within a suitable range. When the data is captured, it can be sent from measurement board  102  to be stored for example in computer RAM forming part of device  103 . The signal such as shown in FIG. 10 is analysed by, for example, applying a smoothing algorithm to the raw data to remove high frequency noise. 
     Within a given set or batch of captured data, the system detects a pulse signal S by an increasing leading edge over time. A minimum number of positive going sample values are required to trigger the search for a corresponding negative going edge thereby to detect a pulse signal S. A potential pulse is rejected if a minimum number of negative going samples is not detected after passing through a peak position. The system measures the width of the signal as the point half way along the leading edge to a point on a trailing edge where the voltage becomes less than the starting point on the leading edge. Knowing the expected particle flow rates, it is possible to detect falling fluid flow through the orifice by an increasing pulse width. This can be indicative of a partial blockage of orifice  63 . However, if the pulse width is too narrow, this data is rejected as noise rather than a signal. The pulse height of the signal S can be measured between a net baseline level in the noise N, adjacent signal S and mean or average, peak value of the signal S. As an alternative, the peak height can be determined simply from the value at the first positive going point in a detected pulse signals and taking the difference between this value and that at the pulse maximum. The pulse area is calculated by integration of signal S and is stored both for a batch and an accumulative histogram for the sample. 
     Occasionally, two particles pass through orifice  63  at the same time. This can result in a single pulse representative of the volume of both particles, however, sometimes both pulses are resolvable from one another since a first peak is observed followed by a negative going pulse which does not reach the base line voltage before a second positive going pulse due to the second particle occurs. These incidences can be referred to as primary and secondary coincidence respectively. Preferably, the sample concentration of say a million particles per ml is such that these coincidences very rarely occur, however, pulses from secondary coincidence can still be analysed and form part of the data by extrapolating along the negative going edge of the first pulse to determine a pulse width and also by extrapolating the positive going edge of the second pulse also to determine a pulse width. The initial value at the leading edge of the first pulse can be used in the determination of the height of the second pulse. 
     For each signal detected in a batch the pulse width and pulse height are stored and added to data for that batch. If it is determined that the batch of data is good, checks carried out are described later, the batch data is added to a cumulative pulse height histogram, which is indicative of the particle volume distribution for the sample, and the cumulative pulse width histogram. In this example each batch of data represents the signals over 32 ms of data capture. Also the number of pulses detected is stored as a current batch value and total for all batches for a given sample (i.e. series of measurements). A further store of noise can also be conducted of say any region of noise not containing a pulse signal S for a given a set of captured data. A current value derived from that before and after the batch of data is stored as a mean value of the two measurements. The current value is output at PCRT of the circuit shown in FIG. 9, or line  104  of amplifier board  92  shown in FIG.  10 . The level of vacuum in reservoir  76  is also measured for each batch. Batch data is added to overall sample data to provide cumulative sample histograms. 
     Typically, measurements of a sample are made over a twenty to thirty second time period during which the sample is caused to flow through orifice  63 , preferably a particle at a time. The flow can be maintained by repeated evacuation of vacuum reservoir  64  using pump  62  so as to retain an under pressure in the reservoir which draws sample from chamber  52  through to chamber  58 , ie. due to the over pressure of atmospheric pressure at inlet  16  to chamber  52 . For example, three successive evacuations of chamber  64  to a preset pressure might be necessary in a thirty second measurement period, depending on how often a deblocking sequence, described later, is conducted. 
     Under block conditions, the impedance between electrodes  82  and  84  varies significantly and this affects the gain, of what could be considered the orifice  63 , or amplifier stage IC 10  and so on. Under these conditions, a pulse signal S can approach that of a square wave. This can be monitored by analysing a detected pulse signal S from the time difference between the signal increasing through two distinct and predetermined voltage thresholds above the DC off-set level, and the time for the signal to return back through the two predetermined voltage levels. If the time difference between passing through the lower levels and the upper levels is substantially equal and non-zero then it is indicative of a block having occurred at orifice  63 . Additionally, where a square wave signal is followed by a recovery curve which has a characteristic recovery rate determined by the electronic circuitry and which is positive going, in the sense of signal S shown in FIG. 12, then this signal is indicative of a very large particle in the vicinity of orifice  63 . The pulse is designated a rejected pulse. 
     The background noise N is also monitored. By analysing the frequency domain of the noise, following Fourier transformation, it is possible to detect a partial block since the gain of the amplifier is affected and anomalous frequency amplification can be seen. If a factor of say two difference in amplitude is seen in any frequency component then the data can be rejected. Accordingly, the comparison between background noise and an average background noise can indicate partial or complete blockage of orifice  63 . 
     To check if a given batch of data is acceptable, a first check can be the number pulses observed in the period of the data capture. If the number of pules vary significantly in comparison to the mean number of pulses per batch, say by greater than or less than 50%, or alternatively one standard deviation from the mean, the data can be rejected. Also, comparison of the mean current value at the start and finish of batch data capture with the start current value can be made. If the difference is greater than say +/−20% then the batch data can be rejected. Additionally, the mean current value compared to the mean current value for the previous batch of data can be checked. If the difference is more than say an increase of 10% or a decrease of 5%, then the second batch data can be rejected. Additionally, comparison of the noise with static limits can be made. For example, using the Fourier analysis if a given frequency component varies by a factor of two in comparison with the mean, then this can be taken as defective data. Comparison of the mean pulse width for a batch of data with the cumulative mean pulse width can be made. If the discrepancy is greater than say 10% then the batch of data can be rejected assuming that a significant number of pulses is detected per batch, say at least 30 and preferably 100 pulses per batch. Additionally, the number of rejected pulses, or square wave pulses is monitored, data can be rejected when observing either one or more rejected pulses in a given batch. In the event of any of these events occurring which leads to data rejection, a deblock sequence is preferably initiated. 
     Preferably, the deblock sequence is initiated by turning off the DC voltage on electrode  82  and applying a pulse sequence which can be the calibration pulse sequence. Accordingly, an ultrasonic vibration, in the frequency range 15 to 20 kHz, is set up within the electrolyte. Chamber  58  is also primed by operating pump  46 , opening valve  56 , whilst valve  74  is also open. The priming sequence is terminated after a short time period of say 100 milliseconds, and the calibration or ultrasonic pulses are also turned off. The large DC voltage is reapplied to electrode  84  and a time delay of say one second is allowed to elapse to stabilize fluid flow. The data measurement sequence is then recommenced as the measurement board is re-initiated, again the attenuator board is adjusted and an initial current value recorded. 
     Preferably, the data received immediately preceding a deblock sequence is subtracted from cumulative data and rejected as being inconsistent. 
     The measurement sequence is repeated until the required quantity of fluid has passed through the orifice. This is determined knowing the size of orifice  63 , the flow rate from pulse widths and the duration of measurement. Measurement of a sample can also be stopped when the required number of particles within a set range has been measured, or a fatal error occurs and is not rectified such as the orifice current changing and not returning to the value of the start current, the diluent reservoir becoming empty, the data repetition becoming inconsistent, or the base line noise level rising above an acceptable static level. 
     Upon completion of a measurement sequence, the sample is evacuated from chamber  52  by opening the valve  80 . The sample side of the chamber  52 , can then be cleaned through a number of rinse cycles by opening valve  51  and pumping at pump  46 . 
     Preferably, an intelligent washing sequence is performed whereby the concentration of sample particles from a previous sample is monitored i.e. from the number of particle signals detected in a known measurement volume. The apparatus can be calibrated to determine how many rinses are required to clean the chambers following a given concentration of sample.

Technology Category: g