DE2261695C2 - Device for rapid analysis and sorting of small particles - Google Patents

Description

The invention relates to a device according to the preamble of claim 1.

In particular, such a device can rapidly and automatically measure and analyze the volume, shape and fluorescence of biological cells suspended in a continuously flowing fluid to determine whether the cells appear normal or abnormal, with the cells identified as abnormal being physically separated from the normal cells. In the field of cytology, it is becoming increasingly necessary to provide for automatic cell analysis and cell differentiation. Currently, the examination of cytological material, for example to detect cancerous or malignant cells, is carried out by two or more stages of examination. Initially, the cell samples are visually examined by an observer to pick out the samples containing abnormal cells. These samples are then set aside for later examination by a trained cytotechnologist or pathologist who will then make a final judgment as to whether the cells are in fact cancerous. Although this method currently works satisfactorily, it has a number of disadvantages. First, it is very time consuming and requires a lot of technician time, so that it is also expensive; Furthermore, it does not provide quantitative results, since the criterion of abnormality used is largely subjective. Because of the time and costs involved, the known method cannot be easily applied to large numbers. Moreover, many, possibly most, of the samples submitted to the medical laboratory are Cell samples are normal. For example, in cytological testing for uterine cancer, 98% of the women examined do not have cancer. The result of this is that - especially when larger populations are being screened for cancer - the level of attention and interest of those performing the screening is reduced. Over time, this means that a test becomes less quantitative and more expensive as staff turnover increases.

Many of these disadvantages can be overcome by the use of flow system techniques in cell analysis to perform screening. Flow system analysis allows the observation of individual cells as they flow in suspension one after the other through a small volume of study. Large numbers of cells can be observed in short periods of time, and rapid automated screening procedures are possible. Light absorption, fluorescence, scattering, or volume of particles observed are common parameters. Various claims are made in the literature that these parameters have been observed quantitatively; a major difficulty is that a single parameter is often insufficient to quantitatively distinguish between normal and abnormal cells. Multiparameter analysis increases the ability to distinguish between different types of cells. Because the majority of cells examined are normal, means of sorting out the abnormal cells from the normal cells are highly desirable, so that the sample generated for subsequent examination contains a preponderance of cells considered abnormal. These various considerations and the current state of the art are described in detail in Part A of the following reference: "Automated Cytology: A Symposium by Correspondence", Acta Cytologica, Vol. 15, Nos. 1-3 (1971). In US Patent 33 80 584, one of the inventors of the present invention has described an apparatus having the features of the preamble of claim 1 for sorting small particles suspended in a fluid. Sorting is carried out according to a selected parameter which may be, for example, size, volume, presence of radioactivity, color, fluorescence, light absorption or any other quantity which can be converted into an electrical quantity. However, the particle separation apparatus according to US Patent 33 80 584 is based on the measurement of a single parameter and not on a multi-parameter measurement.

Only one device is known for sorting abnormal cells from large populations of normal cells based on multiparameter analysis. Kamentsky and Melamed in "Science", volume 156, page 1364 (1967) describe a spectrophotometric cell sorter that separates cells with predetermined optical properties from large populations of cells in suspension. Sorting is done on the basis of multiple optical measurements, and the separation system uses fluid switching principles that were used in computer design around 1964 (Fluidios). This spectrophotometric cell sorter operates relatively slowly and is not able to produce a sample consisting primarily of cells that are to be further studied. For example, even with the best efforts, this cell sorting device is not capable of producing final concentrations of selected cells (i.e. cells abnormal with respect to predetermined norms of normality) of approximately 1:5 from initial concentrations in the range of 1:10,000.

It is known that the performance of cell volume sensing instruments based on the principle of the Coulter counter - in which a cell changes the impedance of a narrow orifice as it passes through an orifice - can be improved if the cell suspension is surrounded by a coaxial flow of cell-free liquid as it passes through the orifice. For example, Merrill et al. in Rev. Sci. Instru., Volume 42, page 1157 (1971) describe a cell volume analyzer with coaxial flow of the cell suspension within an envelope of cell-free solution through the sensing orifice. However, this device is not a cell sorting device and operates as a single parameter analyzer. Although Merrill et al. suggest that this device can also be used for multi-parameter analysis, there is no indication in the prior art that this has actually been done.

The invention is based on the object of designing a device according to the preamble of claim 1 in such a way that the rapid and automatic analysis and sorting of small particles is made possible, namely on the basis of certain preselected properties or combinations of these properties.

This object is achieved with a device of the type mentioned, which has the features of the characterising part of claim 1.

The subject matter of the subclaims are preferred embodiments of the device according to the invention.

The device according to the invention is a further development of the device shown in US Patent 33 80 584 and allows particle separation on the basis of multi-parameter analysis. The device can be used in particular in the analysis and sorting of biological cells.

In one embodiment of the apparatus useful for sorting abnormal (malignant) cells from normal cells, cellular volume, small angle light scattering and fluorescence are measured for each cell and compared to predetermined standards, separating cells not conforming to these standards from cells conforming to these standards. Cell samples stained with an appropriate fluorescent dye are diluted and suspended in a physiological saline solution and introduced into a flow chamber on the axis of a moving stream of saline solution, the latter acting as a sheath to confine the cell flow to the central axis of the system. Within the chamber, cells flow sequentially through an orifice which serves as a Coulter volume sensor and where cell volume is measured electronically. Next, the cells flowing in suspension in the saline solution intersect an argon ion laser beam. The individual cells scatter the light and the dye bound to the cells is excited to fluoresce. The scattered light provides quantitative information about cell size and shape, while fluorescence provides a quantitative measurement of all cell components to which a fluorescent dye is bound, for example, DNA content can be indicated. Small angle light scattering is measured in the forward direction and fluorescence is measured perpendicular to the cell flow and laser beam. After passing through the laser beam, the cell suspension is jetted out into the air through a coaxially aligned nozzle at the exit end of the flow chamber. A piezoelectric crystal mechanically coupled to the flow chamber is used to produce uniform droplets by periodically dispersing the exiting fluid jet. Most cells are effectively separated into individual droplets, although not all droplets contain cells, and although certain droplets may contain two or more cells. Selected droplets containing cells are electrically charged and then deflected by a static electric field into a separate vessel. An oscilloscope monitors individual signal pulses while a multi-channel pulse height analyzer, printer, and plotter generate and record pulse amplitude distributions representing cell volume, light scattering, and fluorescence, or combinations of these properties. A pulse generator, triggered by a single-channel pulse height analyzer, having a variable delay, generates droplet charging pulses that are delayed to allow the sorted cell to travel from the sensing zone to the point of droplet formation and charging.

Preferred embodiments of the invention emerge in particular from the subclaims.

In the following, the invention is explained in more detail using embodiments and the drawing. The drawing shows

Fig. 1 is a block diagram illustrating how the device according to the invention can be used in a cancer screening program;

Fig. 2 is a simplified view of the apparatus of the invention showing the flow chamber and the loading and deflection plates used for particle sorting;

Fig. 3 is an enlarged, simplified sectional view of the sensing portion of the flow chamber;

Fig. 4 is a detailed cross-sectional view of the flow chamber usable in a preferred embodiment of the invention;

Fig. 5 is a block diagram of the optical and electrical elements of a preferred embodiment of the invention;

Fig. 6 shows a part of a logic and circuit block diagram for the multi-parameter signal processing unit indicated in Fig. 5;

Fig. 7 is a continuation of the diagram of Fig. 6.

The device according to the invention can be readily used for rapid and automatic multi-parameter analysis and sorting of various types of particles. The size of the particles to be analyzed is limited by the size of the Coulter volumetric opening. Furthermore, the particles to be analyzed and sorted by the device according to the invention must be amenable to analysis on the basis of their physical and chemical properties.

The figures shown here relate to an embodiment of the invention which is useful for analyzing and sorting abnormal cells from normal cells in a cytological examination program for the determination of cervical cancer. The scheme is shown in Fig. 1. First, the cell samples are prepared for flow system analysis by appropriate dilution, treatment to avoid clumping, staining with fluorescent dyes, etc. This preparation is done according to the particular type of automated analysis to be used. In this particular setup, the following cell parameters are measured: cell volume, low angle light scattering and fluorescence. The fluorescence measurements depend on the use of biochemical specific dyes. The sensing or measuring devices provided for performing these particular measurements are compatible with each other and also with the electronic sorting of the cells. The electronic sorting method used is similar to that described in U.S. Patent 3,380,584. As each cell is analyzed, a signal from each measuring device is delivered to a multi-parameter signal processing unit where it is processed and compared to predetermined criteria of abnormality. Thus, while the cell is still in the vicinity of the measurement zone, the signals from the measuring devices representing the measured cell properties are processed into ratios, overlap regions, etc. that most effectively describe abnormal cells. The processed signals are electronically compared to specified standard values (norms), and the corresponding cell is classified as either normal, abnormal, or doubtful. The time required for signal processing and sorting decisions after the signals are received is on the order of 25 microseconds. If a cell is classified as abnormal or doubtful, a signal is generated which causes a droplet containing that cell to be deflected from the droplets containing normal cells. The analysis results for that cell can be stored separately from the data for normal cells in the sample. The rejected, abnormal or doubtful cells are additionally stained and kept ready for visual examination by a cytologist. To assist the cytologist in evaluating the sorted cells, distributions of the various measured cell properties or combinations of the properties of the entire sample or only of the abnormal cells to be examined are available from the data processing storage device. available. The device according to the invention produces both printouts and an oscilloscope display of the data.

The specific embodiment described here is concerned with multi-parameter analysis of the volume, fluorescence and small angle light scattering of individual cells, but it will be apparent to those skilled in the art that the analysis and sorting methods and devices described here are applicable to other types of high speed sensing or measurement, and that the electronic and mechanical components of the embodiment can be readily modified to allow the measurement of other parameters. For example, with the flow chamber described here, small angle light scattering measurement can be replaced by measurement devices that detect light absorption or fluorescence at a different wavelength.

Description of the preferred embodiment.

2 and 3 illustrate the basic flow system and sensing or measuring zone of the device of the invention. A suitably prepared cell sample is introduced as a continuously flowing suspension into the flow chamber 3 through a sample inlet tube 1 from a pressurized reservoir 72. Within the chamber 3 , the tube 1 is centered and extends partially through a larger tube (first sheath fluid tube) 19 which tapers to a nozzle 22 at its lower end. A continuous flow of cell-free fluid - known as sheath fluid - is introduced into the tube 19 through a sheath fluid inlet tube 18 which communicates with a pressurized reservoir 70 , the sheath fluid flowing coaxially - see reference numeral 20 - around the tube 1 . As the cell stream exits tube 1 , it is reduced in diameter 17 as it maintains the velocity of the sheath fluid. The relative velocities and flow rates are determined by a differential pressure regulator system. It is necessary that the coaxial flow of the sheath fluid and the cells in suspension be substantially laminar so that turbulence effects are avoided as the sheath fluid and cell suspension pass through the volume sensing orifice 21 in nozzle 22. The pressure differential between the sheath fluid 20 and the cell stream is adjusted so that a cell stream 28 passes through the orifice 21 which has a diameter such that most of the cells pass through individually. The orifice 21 serves as a Coulter volume measuring orifice in which the impedance is varied according to the volume of the cell passing through. The laminar flowing sheath fluid not only controls the magnitude of the cell flow passing through the opening 21 , but also serves to center it within the opening so that the edge effects of the electric field influencing the volume measurement are significantly reduced.

After leaving the opening 21, the cell stream 28 intersects a beam 4 of intense light. The flow chamber 3 is further provided with an inlet opening 35 through which this beam can be focused onto the cell stream. Openings 36 (one of which is not shown in Fig. 2) serve as outlets for the fluorescence 9 and light scatter 11 produced as a result of the interaction 16 of the light with a single cell as the cells flow in sequence in the cell stream 28. The light beam 4 intersects the cell stream 28 at an angle of approximately 90°. Fluorescence is emitted in all directions but is only measured in a cone 9 extending at right angles to the plane of intersection of the cell stream 28 and light beam 4. These 90° angles are critical in that they simplify the required optical measurements.

The cell stream 28 is jetted out of the flow chamber 3 through the exit nozzle 26. It is essential that the orifice 21 be properly aligned with the nozzle 26. Misalignment can interfere with the optical measurements due to turbulence, and can affect the precise timing by which measurements associated with a particular cell are translated into a sorting signal because the cells lose their equal sequential spacing within the cell stream 28. It is also necessary that the cell stream and its surrounding sheath fluid exit the flow chamber 3 at a sufficiently high velocity to form a jet 10. Because of the pressure drop associated with the orifice 21 , an additional source of sheath fluid is required to generate sufficient pressure in the zone 30 to form an adequate jet 10. This second sheath fluid input 25 is via a pipe connected to the sheath fluid input pipe 12 (second sheath fluid pipe) 54 . The tube 12 is in turn connected to a pressurized sheath fluid reservoir 71. The inlet 25 is sufficiently far from the cell stream 28 so that other than increasing the velocity through the nozzle 26 , no effect is exerted on the cell flow. The sheath fluid inlet 25 has the additional advantage that it allows the flow chamber to be flushed to remove accumulated gases. Since the volume measurement involves the use of an electrical current, the liquids present in the flow chamber 3 have a tendency to electrolytic dissociation. The dimensions of the flow chamber are sufficiently large so that gas bubbles resulting from such dissociation can rise to the top of the flow chamber where they are temporarily stored without disturbing the optical or electrical measurement. However, it is desirable to have some means to periodically flush any accumulated gases from the system. This is easily achieved by the inlet tube 12 . When flushing is required, a valve in the flushing outlet pipe 2 is opened and additional sheath fluid is supplied to the flow chamber 3 through inlet pipe 12 .

A vibration device (vibration means) 31 is coupled to the flow chamber 3 by a coupling rod 32. The vibrations (oscillations) imposed on the flow chamber 3 generate very small disturbances or swellings 29 on the jet 10. By generating these disturbances at a correct frequency determined by the jet diameter and the speed, they grow in amplitude due to the surface tension until the jet 10 is divided into droplets 13 that have the same distance and size. In this way, the cells in suspension are broken down into liquid droplets. The manner in which the vibrating device 31 is coupled to the flow chamber 3 is not critical, except that the vibration frequency must be kept relatively constant. Typically a piezoelectric crystal is used as the vibration or oscillation source, but other means may be used.

The droplets 13 thus produced pass between the charging electrodes 5 where those droplets which are determined to contain abnormal cells based on analysis of the volume, light scattering and fluorescence of each cell are charged. The droplets containing normal cells are not charged. This particular order is provided because it is assumed that most droplets contain normal cells, so it is easiest to charge only the droplets containing abnormal cells. However, it is also possible to use the exact opposite procedure. The droplets then pass through an electrostatic field between the deflection plates 8 . Under the influence of this field, the charged particles 14 are deflected into different vessels 7 than the uncharged particles 15 .

Fig. 4 is a detailed cross-section of a flow chamber suitable for use in the present invention. The suspended particles flow into the chamber through tube 1. This tube also serves as an electrode for the Coulter volumeter and may be made of any suitable conductive material. In the preferred embodiment, this tube is made of a platinum-rhodium alloy to avoid corrosion problems arising from the use of a physiological saline solution which is used both as a carrier fluid for the biological cells and as a sheath fluid surrounding the carrier fluid. The tube 1 is coaxially aligned within a larger tube 19 by means of a guide star 16 and is provided with a nozzle 47. This nozzle may be made of platinum, but it is not necessary that it be. A non-corrosive material such as a suitable plastic may be used as well. The guide star 46 and nozzle 47 may be combined and formed of the same material. The tube 19 terminates in a nozzle 22 in which the Coulter volumetric orifice 21 is located. The tube 19 may be made of any non-conductive material such as glass. In the preferred embodiment, the volumetric orifice 21 is located in a sapphire insert 34 which is connected to a glass tube 19. Sapphire is used because it is readily available, but the insert 34 may be made of any other suitable non-conductive material in which a suitable orifice can be formed.

Through an inlet tube 18 the sheath liquid is introduced into tube 19. The sheath liquid also enters cuvette 23 through inlet tube 12 and purge tube (second sheath liquid tube) 54. Purge tube 54 also serves as the second electrode for the Coulter volumeter so both tubes 54 and 12 must be made of a suitable conductive material. In the preferred embodiment tube 12 is made of platinum and tube 54 is made of a platinum-rhodium alloy. Purge outlet tube 2 connected to the inner zone 30 of cuvette 23 removes any gases generated in zone 30. In the preferred embodiment outlet tube 2 is provided with a valve but if desired the sheath liquid can be passed continuously from purge tube 54 through cuvette 23 and out through the outlet tube.

The cuvette 23 is open at the top and may be made of any material which is an insulator and transparent to light at the wavelengths used in the light beam 4. In the preferred embodiment, the cuvette 23 is made of quartz, primarily because a quartz cuvette of the desired size is readily available commercially. Centrally located in the base of the cuvette 23 is an opening 58 through which a nozzle 26 extends. In order to minimize wall effects on the velocity of the cells ejected by the nozzle 26 , the nozzle 26 extends nearly to plane AA in which the optical measurements on the individual cells are made. This extension also allows for easier alignment of the volume measurement opening 21 with the opening 59 in the nozzle 26 .

The cuvette 23 is surrounded by a metal jacket 24. The jacket 24 protects the cuvette 23 and shields the Coulter volumeter from external electronic noise. At the base of the jacket 24 there is a sealing end plate 56 and a nozzle retaining plate 57. The end plate 56 has a circular opening 60 in its center through which the elongated part of the nozzle 26 extends. The nozzle 26 is screwed tightly into the central opening 61 of the end plate 57 .

At the top of the shell 24 is a tightening screw 55 into which a closure cap 52 is screwed. The cuvette 23 is sealingly enclosed by the shell 24 and cap 52 by means of an O-ring 48 disposed in a recess 62 of the end plate 56 and a gasket 51 provided adjacent to the cap 52. The closure cap 52 extends partially into the cuvette 23 to form a recess 63. At the base of the recess 63 is centrally disposed a circular opening 64 through which tube 19 extends. An angled adjustable sealing gland member 44 is screwable into recess 63 until it is flush with the top of the cover cap 52. The member 44 has a conical truncated recess 65 in the center thereof with a lip 66 provided near the upper end. The recess 65 has a circular opening 67 centrally in its base through which the tube 19 passes.

A sealing collar 43 is arranged and secured around the upper end of the tube 19. Above the sealing collar 43 and the tube 19 is a tube connecting and positioning piece 41. The positioning piece 41 has a channel 68 therethrough through which the tube 1 enters the tube 19 ; it is coaxially aligned with the upper part of the tube 19. Sheath liquid inlet tube 18 also enters the positioning piece 41 and sheath liquid enters the tube 19 through channel 69. The inlet tubes 1 and 18 are surrounded by a shield tube 40 which prevents electrical noise from entering the flow chamber through tube 1 or tube 18 . The shielding tube 40 and the positioning piece 41 are held in position at the upper end of the tube 19 by a pressure connection cap 42 which can be screwed onto the sealing ring 43. An O-ring 50 creates a seal between the collar 43 and the positioning piece 41. When the tube 19 is inserted, the recesses 65 and 67 are sealed from the cuvette 23 by O-rings 70. The seal of these O-rings can be achieved by By screwing or unscrewing the packing gland part 44 in or out of the closure cap 52 , the tube 19 can be moved into or out of the cuvette 23 as desired. Four adjusting fixing screws 45 are arranged at equal distances around the packing gland part 44 , although only one of these screws is shown in Fig. 4. These fixing screws 45 form a convenient means for aligning the opening 21 at the end of the tube 19 with the opening 59 in the nozzle 26. As already described above, it is necessary for the correct operation of the device according to the invention that these two openings are precisely aligned.

Figure 5 is a block diagram of the electrical and optical elements of an embodiment of the invention useful for rapidly and automatically analyzing and sorting abnormal cells from normal cells. A piezoelectric crystal coupled to the flow chamber serves as a source of vibration or oscillation to produce droplets of the desired frequency. The light source used in this embodiment is an argon ion laser appropriately focused to intersect the cell stream in the flow chamber. The light beam entering the flow chamber typically has an elliptical cross-section to assist in analyzing cell structure by means of the resulting light scattering and fluorescence. The laser beam is optically shaped to have an elliptical cross-section at the intersection of the laser beam and the cell stream. The elliptical cross-section facilitates operation (alignment), increases signal strength with improved resolution, and allows characterization of cell structure (nucleus to cytoplasm ratio) and duplicate detection. Doubles are two cells that pass through the flow chamber in contact with each other. To the volume measuring device they appear as one abnormally large cell. In order not to obtain false data from the measuring devices, some precaution must therefore be taken.

An argon ion laser is used as a light source because cancer cells normally contain much more DNA than normal cells, and the fluorescence of an excited Feulgen dye biochemically bound to the DNA in the cell is a quantitative indication of the DNA present in the cell. The argon ion laser emits light at a wavelength suitable for exciting this dye to fluorescence. The fluorescence pulses coming from the flow chamber as a result of the interaction of the light beam with the cells are focused by a lens system onto a pinhole and then onto a photomultiplier tube. The signal from this photomultiplier tube is amplified and fed to a multiparameter signal processing unit.

Theory suggests that small angle light scattering (at angles between 0.5 and 2.0°) by spherical particles of 5-20 microns diameter is nearly proportional to volume. Since most mammalian cells have diameters in this range, light scattering is a promising means of obtaining size and structural information for individual cells at high speed. In the preferred embodiment, light scattered by the cells between 0.5 and 2.0° is passed through a converging lens system onto a photodiode. Light scattered by less than 0.5° is passed into a beam annihilator. In this way, the photodiode is not overloaded by light that has not interacted with the cell current. The photodiode signal is amplified and also fed into the multiparameter signal processing unit.

The signal generated by the passage of individual cells through the Coulter volumetric aperture has the advantage that it is already electrical in nature, so that it only needs to be amplified and fed into the multi-parameter signal processing unit.

As the name suggests, the multi-parameter signal processing unit processes these input signals, compares them to certain predetermined standards, and then produces three types of output signals. One signal is fed to a multi-channel pulse height analyzer, which in turn produces digital printouts, a pulse height analyzer display, and histograms of the data obtained from the multi-parameter signal processing. The signals from the processing unit can also be monitored directly by an oscilloscope display. Finally, an output of the processing unit is passed through a single-channel analyzer, and through separation logic devices and a droplet charging generator to generate droplet charging pulses used to separate selected cells from the cell stream. It will be appreciated that time delay means are used in conjunction with the multi-parameter signal processing unit to coordinate all sense or measurement signals with a particular cell.

The signals received by the multi-parameter processing unit can be processed in different ways to modify their dependence on the measured property.

For example, a signal proportional to cell volume (r ³ ) can be processed to be linearly proportional to cell radius (r) or area (r ² ). Because only one data item is generated for each measuring device and for each cell, the amount of data to be processed is small and the electronics requirements are not great. By using a two-dimensional pulse height analyzer, a two-parameter frequency distribution of the cells can be obtained. Three- or multi-parameter analysis requires the data capacity of a small computer. As an alternative to storing all the information, logical constraints can be placed on the analysis scheme, thus reducing the electronics requirements. For example, if the fluorescence distribution of all cells within a certain volume range is desired, this can be obtained by analyzing the fluorescence of only those cells that produce a volume signal corresponding to the desired range. In a similar way, the volume of cells within a certain fluorescence range can be obtained. When biologically useful information is generated, analysis is possible on the basis of three or more such logical requirements.

Alternatively, the processed signals from multiple measurement devices can be combined as ratios (or sums, differences, etc.) and the frequency distribution of the combination can be determined among a population. For example, by using RNA-specific fluorescence staining and by fluorescence and volume measuring devices, a ratio of the processed signals is formed to obtain a distribution of RNA density among a population of cells. Similarly, a type of nucleus-to-cytoplasmic ratio is obtained by using a nucleus-specific fluorescent stain to give a nuclear volume measurement and a total cell volume measurement by scattered light or the Coulter measuring device.

A logic and circuit block diagram for a multi-parameter signal processing unit useful for analyzing and sorting abnormal cells from normal cells is shown in Figures 6 and 7. The multi-parameter signal processing unit is the central electronic processing unit for single-parameter analysis, ratio calculation, series or array analysis and subsequent cell sorting. Basically, the signal processing unit serves as a central analog electronic computing interface between the measuring devices, the multi-channel pulse height analyzer and the cell separation controller (single-channel analyzer; separation logic; droplet loading generator). The signal processing unit also produces x and y outputs for a two-parameter pulse height analyzer. Amplified signal pulses (0.4 to 8.0 volts) from the Coulter or cell volume (CV), light scattering (LS) and fluorescence (FL) measuring devices are fed directly into the processing unit. The unit has separate inputs for the volume, light scattering and fluorescence signals. If desired, a fluorescence signal with a second wavelength, for example red, can be substituted for the light scattering input signal or a wide angle light scattering signal can be substituted for the fluorescence input signal.

The signal processing unit serves as a controlled signal peak measure and hold device capable of both single processing and dual processing of the measurement signals (sensing signals). The processing unit is divided into three sections: input conditions, signal processing and output control. The input conditions section (shown in Fig. 6) consists of mode and CV-FL/LS delay selector switches. The eight-position mode switch allows single parameter cell analysis, ie CV, LS and FL, and two parameter analysis of cells, ie CV and FL, CV and no FL, CV and LS, LS and FL and finally LS and no FL. Since the Coulter volume signal arrives before both the light scatter and fluorescence signals, a variable (0 to 190 microseconds, in 10 microsecond increments) CV-FL/LS delay switch is used to set the proper CV to FL/LS signal delay. This delay is on the order of 170 microseconds. The delay only needs to be used in the two-parameter analysis mode when the Coulter volume must be analyzed.

The signal processing section consists of ratio and series (input and analysis) analysis selector switches (see Fig. 7). A six-position ratio selector switch allows the calculation of the following ratios: CV/FL, CV/LS, FL/CV, FL/LS, LS/CV and LS/FL. It is essential that the mode and ratio selector switches match, for example the mode selector is in the LS and FL positions and the ratio selector is in either the LS/FL or FL/LS ratio position. It is also important that the CV to FL/LS signal delay is used when calculating ratios containing CV. The series or sequential analysis section consists of input and analysis selector switches (each with four positions). The series analysis input selector switch selects either CV, FL, LS or ratio signals fed to an external single-channel pulse height analyzer (SCA series). When the signal amplitude falls within a variable width preset on the SCA window (0.4 to 8.0 volts), an SCA trigger pulse is generated and fed back to the signal processing unit, which gates the series analysis linear gate to analyze either the CV, FL, LS or ratio signal as determined by the series analysis analysis selector switch. Both the series analysis input and analysis selector positions (CV, FL, LS and ratio) must match the CV FL/LS delay mode and ratio selector switches whenever required, for example, series analysis input CV, series analysis analysis FL, mode CV and FL, CV FL/LS delay ~180 µsec and ratio off.

The output control section (see Fig. 7) consists of a pulse height analyzer (PHA) input, a separator input, and two-parameter analyzer input selectors. The PHA input can select individual parameters (CV, LS, or FL), ratios, series analysis input, and analysis parameters for external routing to a multi-channel pulse height analyzer, whereas the separator input selector can feed individual parameters, ratios, or series analysis analyzer signals to the separator controller. The two-parameter pulse height analyzer selector produces the following output quantities: CV _x -FL _y , CV _x -LS _y , LS _x -FL _y , CV _x -ratio _y , FL _x -ratio _y and LS _x -ratio _y , where the subscripts x and y refer to the x and y axes of the two-parameter PHA. By swapping the x and y axis inputs, the above quantities can be reversed.

Sequence of workflow

The cell populations to be studied are first stained (fluorescent Feulgen stains, etc.) and placed in an aqueous suspension such as normal saline. Bound and unbound cells can be measured. Before the cell suspension is placed in the cell reservoir, it is filtered through a 60-70 micron nylon mesh screen to remove large contaminants and clumps. The electronics are already in standby mode, the system is pressurized, and the laser is on. Before cell measurements, the system is aligned and adjusted. If cell sorting is required, the droplet generator oscillator amplifier that electrically drives the piezoelectric crystal or equivalent transducer must be turned on. Droplet formation is checked by illuminating the emerging liquid jet near the flow chamber with a stroboscopic light or equivalent light source. The stroboscopic light is flashed synchronously with the oscillator frequency. Droplet formation can then be observed using a microscope. For a given nozzle diameter and flow velocity, droplet formation can be changed by varying the voltage and frequency applied to the piezoelectric crystal. Typical values are 15 volts _rms (sinusoidal) at 40 to 50 kHz. The droplet charging electrode is placed on either side of the point of droplet formation (separation) approximately 0.79 cm below the flow chamber to ensure maximum droplet charging. Typical charging pulses are 50 volts at 100 to 200 microseconds. The electrostatic deflection plates are placed 5.0 cm-7.6 cm below the flow chamber and are spaced 1.27 cm to 1.9 cm apart. A differential of 15 kV DC is typically applied to the deflection plates. A sample collection cup or other suitable collection system is placed 20 cm to 22.5 cm from the flow chamber exit side, slightly offset from the (uncharged) main jet, so that only the (charged) deflected droplets are collected. If sorting of cells is not desired, then the above procedure can be omitted.

The suspended cells are placed in a pressurized (161.34 Pascal) cell reservoir. Pressurized sheath fluid # 1 (165.47 Pascal) and sheath fluid # 2 (137.9 Pascal) are turned on and proper droplet formation is achieved if sorting is desired. Sheath fluid # 1 has a flow rate of 0.3 milliliters per minute with no cell flow. The flow rate of sheath fluid # 2 is approximately 3.9 milliliters per minute. The total flow rate through the 86 micron diameter exit nozzle is thus 4.2 milliliters per minute. For a typical cell stream diameter of approximately 20 microns, the cell reservoir pressure of 160.63 Pascals corresponds to a cell flow rate of approximately 0.08 ml/min. The cell stream flow rate can easily be varied from 0 to 0.3 ml/min. (100%) by adjusting the cell reservoir pressure with respect to the reservoir pressure (±1.379 Pascals) for shell # 2 , with the reservoir pressure for shell # 1 remaining fixed. Normally, the pressure for shell # 1 remains fixed relative to the pressure for shell # 2 , but can be changed if desired. Increasing the pressure for shell # 1 relative to shell # 2 decreases the transit time for the cells through the flow chamber. When the sample on/off valve is turned on, cells pass from the cell reservoir through the sample inlet tube into the flow chamber. The inlet tube serves as a Coulter volume signal electrode. From the inlet tube, cells pass through the volumetric orifice (75 micron diameter orifice) where the cell volume is measured. Other orifice sizes may be used. A particle-free sheath (sheath # 1 ) flows coaxially around the sample inlet tube and serves to center the cell stream as it passes through the volume measurement orifice, thus improving the resolution of cell volume and fluorescence light scattering measurements. Typical orifice DC currents from the volume signal electrode through the orifice can be adjusted between 0.05 and 1.0 mA. The orifice current and gain can be adjusted to produce volume signal pulses (0.4 to 8.0 volts) which in turn are fed to the CV input of the multiparameter signal processing unit. Typical volume signal rise time is approximately 20 microseconds with pulse widths of 40 microseconds.

After energizing the volumetric orifice, the cells intersect the laser beam, producing light scattering and fluorescence. Typical delay times between the start of the Coulter volumetric measurement and the fluorescence/light scattering pulse are on the order of 160 to 180 microseconds. The electro-optical fluorescence and light scattering pulses are amplified (0.4 to 8.0 volts) and applied to the appropriate inputs on the signal processing unit. Typical rise times are on the order of 1 to 2 microseconds, with pulse widths approximately 5 microseconds. A second particle-free sheath fluid (sheath # 2 ) of normal saline solution is used to reduce the effect of the pressure drop produced by the Coulter orifice.

Once the properties of the cell are measured, it exits the flow chamber via the exit nozzle in a liquid droplet which can subsequently be separated. The approximate delay time between cell measurement and droplet formation is on the order of 1400 microseconds.

The signals from the volume, light scattering and fluorescence measuring devices and amplifiers are thus fed into the multi-parameter signal processing unit for subsequent analysis and processing. The processing unit serves as an interface between the multi-channel pulse height analyzer, the cell separation logic and the droplet charging generator, as well as the two-parameter pulse height analyzer (not shown in Fig. 5). The signal processing unit must be designed in the correct manner - as mentioned above - depending on the requirements of each test run.

In a typical attempt to analyze and sort out abnormal cells from a given population mixture, a number of different approaches may be useful. The multi-channel pulse height analyzer would first be used to display frequency distribution histograms of individual parameters (volume, light scatter, and fluorescence), ratios of parameters, or possibly serial analysis of parameters, such as analysis of fluorescence for a given cell volume range, etc. The dual or two-parameter analyzer could also be used to analyze various two-parameter frequency distribution histograms as may be required. It is possible to read out abnormalities from various histograms, such as an abnormally large nucleus to cell volume ratio, from the multi-channel PHA display or from the two-parameter PHA display, or from both displays. From this type of information, the cell separation logic droplet loading generator can be enabled to sort out those cells with questionable properties for microscopic examination and identification. The lower and upper threshold levels of the single channel pulse height analyzer (SCA) are set to capture pulse amplitudes (ratios, etc.) of cells with abnormal characteristics. The SCA then triggers the droplet charge generator, which produces a delayed (1400 microseconds) droplet charge pulse (50 volts peak at 100 to 200 microseconds). Once the abnormal characteristics are obtained, it may no longer be necessary to sort out the abnormal cells for examination under a microscope; only further automation of the analysis process is required to promote rapid disease diagnosis.

The optimum performance would be if all cells passed through the volumetric port and the light beam individually, and if only one cell was trapped in each droplet. In practice, this is extremely difficult to achieve. The cells are often spaced widely apart in the cell stream, so that many droplets contain no cells. This is not a particular problem, but if two cells are actually touching (thus forming a doublet) or are so close together that the measuring devices cannot distinguish between them, then the measurement data will indicate abnormal cells and a sorting signal will be provided. If the doublet consists of only two normal cells, as is likely for the normal population of cells, this will result in a dilution of the purity of the sorted sample. Great care in sample preparation can significantly reduce the presence of doublets, and the use of discrimination techniques will reduce erroneous sorting signals due to the presence of such doublets or closely spaced cells, both of which, however, add time and expense to analysis and sorting. For pragmatic reasons, it is often convenient to sort out a certain small percentage of duplicates and cells that are closely adjacent to the abnormal cells. Although this reduces the purity of the sorted sample, it does not hinder the analysis of the sample too much. Typically, 90% or more of the cells are isolated individually in droplets. That is, 90% or more of the droplets containing cells contain only a single cell.

Claims (14)

1. A device for rapid analysis and sorting of small particles based on preselected properties, the device comprising:
a flow chamber ( 3 ) into which a sample containing the particles is introduced in the form of a suspension flowing continuously towards outlet nozzle means,
a device associated with the flow chamber ( 3 ) for determining the preselected properties, and
means for producing uniformly sized droplets which are sufficiently small so that substantially every particle is isolated in a single droplet,
and further comprising means for loading said droplets having the particles with the preselected properties,
and finally, electrical deflection devices are provided to direct the charged droplets away from the uncharged droplets to a separate vessel,
characterized,
that the suspension is introduced into the flow chamber by means of a sample inlet tube ( 1 ) which is surrounded by a first sheath liquid tube ( 19 ),
that the first sheath liquid tube ( 19 ) extends beyond the sample inlet tube ( 1 ) into the flow chamber ( 3 ) and has at its lower end a nozzle ( 22 ) in which a Coulter volume measuring opening ( 21 ) is arranged,
that an observation zone is provided in the flow chamber ( 3 ) adjacent to the Coulter volume measuring opening, which serves to measure further preselected properties of the particles, and
that a second sheath liquid is supplied to this zone via a second sheath liquid pipe ( 54 ). 2. Device according to claim 1, characterized in that the further preselected chemical and physical properties are small angle scattering and/or fluorescence. 3. Device according to claim 2, characterized in that the observation zone has an access opening for the entry of a light beam and multiple observation openings for measuring the optical properties of the particles. 4. Device according to claim 3, characterized in that a laser generates the light beam. 5. Device according to claim 3, characterized in that the first sheath liquid tube ( 19 ) concentrically surrounds the sample inlet tube ( 1 ). 6. Device according to one of claims 1-5, characterized in that the observation zone is surrounded by a reservoir of the sheath liquid which extends above the observation zone. 7. Device according to one of claims 1-6, characterized in that the second sheath liquid tube ( 54 ) extends close to the base of the reservoir, which latter has at its upper end a flushing opening through which gases generated in the reservoir can be flushed out periodically or continuously. 8. Device according to one of claims 1-7, characterized in that the sample inlet tube ( 1 ) serves as an electrode for the Coulter volume measuring opening, and that the second sheath liquid tube ( 54 ) serves as the second electrode for the Coulter volume measuring opening ( 21 ). 9. Device according to one of claims 1-8, characterized by means for controlling the pressures of the liquids entering the flow chamber ( 3 ) through the sample inlet tube ( 1 ), the first sheath liquid tube ( 19 ) and the second sheath liquid tube ( 54 ). 10. Device according to claim 9, characterized in that the pressures are controlled differentially. 11. Device according to one of claims 1-10, characterized in that the oscillation device comprises a piezoelectric crystal which is coupled to the flow chamber ( 3 ) and oscillates at the desired frequency. 12. Apparatus according to any one of claims 1-11, characterized in that the outlet nozzle in the base of the flow chamber ( 3 ) extends into the reservoir, near the plane of the observation zone, means being provided to align the Coulter volume measuring orifice ( 21 ) with an opening ( 59 ) in the nozzle. 13. Apparatus according to claim 12, characterized in that the alignment means comprise a plurality of adjustment screws arranged at a uniform distance around the first sheath liquid tube ( 19 ) outside the flow chamber ( 3 ), whereby the first sheath liquid inlet tube ( 19 ) is rotated about an axis arranged partially within the reservoir. 14. Device according to one or more of claims 4-13, characterized by a generator for generating a first electrical signal proportional to the volume of each cell, by means for measuring the scattering of the laser light and for generating a second electrical signal proportional to the amount of scattering, by means for collecting and measuring the fluorescent light for generating a third electrical signal proportional to the amount of fluorescent light, by means for delaying the first electrical signal and for correlating this signal for each cell with the second and third signals generated by this same cell, and by a comparison device for comparing these signals or combinations of these signals with predetermined value ranges considered normal and for generating an electrical sorting signal when one of the signals or combinations of the signals lies outside the predetermined value range.