APPARATUS FOR CYTOFLUORIMETRIC ANALYSES AND METHODS FOR OPTIMIZING THE CONDITIONING CHAIN Field of the invention

The present invention relates to the field of apparatuses used for cytofluori metric analyses.

State of the art

The cytofluorimetric devices offered by the known art are used in diagnostics in order to measure and characterize cells suspended in a fluid medium.

A great number of cells may be analyzed in a short time by these devices, even 50,000 cells in a few seconds, quantifying many parameters for every single cell, determining the content of DNA, RNA thereof, identifying different cell subtypes, intracellular organelles and also the activity of some enzymes. The main drawback that nowadays still characterizes state-of-the-art cytofluorimetric apparatuses relates especially to the analysis of very rare cells which, because of their reduced number, may often be confused with background noise introduced by the electronic circuitry the cytofluorimetric apparatus is formed by.

The operation of common cytofluorimetric apparatuses is based on the acquisition of two kinds of cell scatter with 3-6 different wavelength fluorescences by means of voluminous appropriate optoelectronic devices.

With reference to Figure 1 , the operation occurs in detail as follows: a monodispersed cell suspension passes through a quartz cuvette with the aid of a fluidic system that allows pumping at different rates. Every cell is hit by a 488 nm wavelength laser beam so that a front and side scatter displaying the same wavelength is generated. A biconvex lens for focusing this scatter and a vertical bar to interrupt the beam generated by the laser are placed in a diametrically opposite position to the laser emitter with respect to said cuvette and is adapted to capture the front scatter. A photomultiplier, indicated by PMT hereinafter, is placed after the bar and serves the function of acquiring and converting the light signal to an electric signal.

An objective for the collection of all of the fluorescent emissions and of the side scatter is positioned at a 90 degree angle with respect to the laser emission. The

objective collimates the beam by directing it to a voluminous and very expensive optical system, which is formed by a sequence of dichroic filters, which may be crossed by certain emissions, for instance having a longer wavelength than a preset value, and reflect others. Specifically, said filters are oriented so as to deviate the filtered beams by about 90 degrees. These are focused by appropriate lenses towards as many PMTs.

Generally, four PMTs are used for as many fluorescences with the addition of two more PMTs for two scatters; therefore, altogether, six PMTs, six focusing lenses, five dichroic filters are required for each fluorescence to be deviated and one deviation for a scatter.

Once the light signals have been acquired as current signals, they must be appropriately conditioned and processed.

Subsequently, the current signal is converted to a voltage signal and sent to a filter, designated Baseline Restoration, by means of which the continuous component is eliminated and the correct offset (designated baseline) is identified, distorted by the electronic noise that could cover up lower intensity signals and lead to interpretation errors such as false negatives.

Said Baseline Restoration filter preferably includes a first current-to-voltage converting stage including a 5 pF feedback capacitor required for high frequency filtering; the second stage consists of an inverting configuration amplifier with a gain equivalent to 2.2 and two feedback diodes required to limit or clip the voltage ensuring a voltage which is not higher than the voltage drop imposed by the conducting diode. The last stage consists of an integrator, the output of which represents the baseline mean value in time. At this point, the signal passes through a preamplification stage with the further addition of small amounts of noise over the signal and the destabilization of the baseline.

Generally, when two fluorophores, for instance centered at 500 and 570 nm and displaying a band width of +/- 40 nm, are used, the fluorescent emissions may be overlapped thus producing a deterioration of the correct spectrum that then needs to be recovered by means of a compensation circuit or a routine within the management software of the cytofluorimetric apparatus.

At this point, to better represent the signals over a wide range of values, it is convenient to carry out a conversion from linear to 4 decade logarithmic (Log amp) and therefore, for this purpose, most of the machines on the market use electronic components, such as logarithmic amplifiers or look up tables. However, also in this case the electronic circuitry employed determines an increase of the background noise.

Subsequently, the signal enters two parallel stages: a trigger stage and a peak detector. The trigger stage serves the function of generating the "start scan" signal, i.e. the correct timing to the analog-digital converter that follows in the conditioning chain. Specifically, it sets the enabling and disabling times of the acquisition board in relation to whether there are cells passing or not. The trigger stage is formed by as many comparators as the signals to be conditioned, therefore 4 fluorescences and 2 scatters for a total of 6 signals. A comparator for each channel implements the function of defining whether a signal is above or below a certain voltage threshold defined by the user, thus producing the "start scan" signal required for the analog- digital conversion.

Said trigger stage also generates additional noise as well as resulting in reply delays. The other stage is instead the peak detector, required for the detection of the extreme points of the fluorescent emissions and of the scatter by the cells. Once the peak has been detected, this is stored in a capacitor and maintained for the period of time required for its acquisition. As well as the peak detector and in parallel thereto, there is an integrator used for computing the appropriate parameters, such as e.g. the area subtended by the half-height curve and its maximum height, designated as FWHM (Full width at half maximum). Finally, the last stage consists of an analog-digital converter (ADC) which, upon enabling by the previous trigger stage, acquires the peaks stored in the capacitors by said peak detector for each channel. In most machines on the market, the ADC has a resolution from 10 bits to 16 bits thus producing a quantization error which may be evaluated respectively from 9.77 mV to 153 μV. The signals produced are therefore stored in a PC which through a

control software represents them on cytograms and dot plots, also computing a great number of statistical parameters required for a following cytofluorimetric reading.

It is obvious that the useful low intensity signals are covered up by the noise and a representation thereof on a cytogram appears in the first half decade, leading to the need to increase the acquisition threshold to be able to avoid it. This also implies the exclusion of low intensity signals, which are very useful in such cases in order to understand how much fluorophore is bound to a cell and specifically in the four-quadrant representation. Furthermore, the conversion by means of integrated circuits follows a determined preset function which may not reflect the modified analysis needs.

To solve such a problem, the known art has introduced a method for the conversion from a linear scale to a logarithmic scale through the use of an eeprom (programmable memory) and an analog/digital converter (ADC). By this method, the signals which are acquired and translated are sent to a 16 bit ADC converter, the outputs of which are connected to the eeprom. The latter serves the function of shifting the input channels (65536 channels) by mapping them on 8 bits (256 channels). However, also in this case electronic components increasing the noise are added. Therefore, it is apparent that, from the perspective of acquiring the light signal, the machines offered by the known art are formed by voluminous and expensive optical assemblies, whereas, from the perspective of the signal conditioning chain, noise is continuously introduced by the electronic circuitry employed with subsequent limitations on the resolution of the measurement and of the analysis carried out.

Summary of the invention

It is the object of the present invention to therefore provide an apparatus for cytofluorimetric analyses characterized by a low cost and a small size.

The present invention therefore suggests to achieve the above mentioned objects by providing an apparatus for cytofluorimetric analyses which, according to claim 1 , is mainly formed by an optical system including: - at least one multi-channel photomultiplier (PMT) (1 ) based on CCD technology

adapted to perform the parallel transduction of at least three different wavelength channels;

- at least one braided optical fiber (4) adapted to detect scatter and fluorescence signals, which conveys them by mixing them on said at least one multi-channel PMT;

- one or more collimating lenses (3) adapted to project said signals on said at least one multi-channel PMT;

- one or more calibrated interferential filters (2), positioned immediately downstream of said collimating lenses, adapted to subdivide the signals of the at least three different channels; and an entirely digital signal conditioning chain.

A further object of the invention is to provide a completely digital signal conditioning chain that may be implemented by means of routine software optimized for said type of apparatus for cytofluorimetric analyses. The present invention therefore suggests to achieve the above said object by providing a completely digital conditioning chain characterized by numerical computing parameters optimized to improve the efficiency of the chain itself and the related methods to obtain them, according to claims X and Y. Advantageously, said apparatus based on said optical system allows the immediate conversion of the signal from analog to digital by means of an A/D conversion board that operates in parallel on all of the output channels from said optical system, so that the remaining conditioning chain operates exclusively in the digital domain allowing to perform analyses which were unknown up until now because of the technological limitations of the apparatuses offered by the known art.

The dependent claims disclose preferred embodiments of the invention.

Brief description of the drawings

Further features and advantages of the invention will become more apparent in light of the detailed description of a preferred though not exclusive embodiment of a cytofluorimetric analysis apparatus, shown by way of non-limitative example with the aid of the accompanying drawings in which: Figure 1 shows a signal conditioning and processing chain;

Figure 2 diagrammatically shows the use of an optical system formed by a multichannel PMT which can, on its own, transduce at least three channels at the same time, one fluorescence channel and two scatter channels, and is combined to a single-channel PMT adapted to detect the front scatter. Figure 3 shows a table in which columns respectively represent the values of the channels on a 16 bit ADC and those on a 8 bit ADC;

Figure 4 respectively shows the limit values of every representation in the linear system and the corresponding logarithmic scale;

Figure 5 shows an interpolation diagram for the scale conversion and for the bit representation.

Detailed description of the invention

The present invention relates to an apparatus for cytofluorimetric analyses that uses an optoelectronic device mainly formed by:

- at least one multi-channel photomultiplier (PMT) (1 ) based on CCD technology adapted to perform the parallel transduction of at least three different wavelength channels;

- at least one braided optical fiber (4) adapted to detect scatter and fluorescence signals, which conveys them by mixing them on said at least one multi-channel PMT; - one or more collimating lenses (3) adapted to project said signals on said at least one multi-channel PMT;

- one or more calibrated interferential filters (2), positioned immediately downstream of said collimating lenses, adapted to subdivide the signals of the at least three different channels; and an entirely digital signal conditioning chain.

Such an optoelectronic device based on the CCD technology allows to acquire at least three signals at the same time, a fluorescence signal and two scatter signals. In a preferred embodiment of the apparatus, said multi-channel PMT can parallelly detect even 16 channels among scatters and fluorescences at the various wavelengths.

Advantageously, the use of an integrated multi-channel PMT allows to eliminate the PMTs adapted to detect the signals irradiated laterally at a 90 degree angle,

with the corresponding lenses, the dichroic mirrors, the band-pass filters, also including the corresponding mechanical allocation means with the corresponding costs and size.

An apparatus based on said multi-channel PMT may include a single-channel PMT for the detection of the front scatter.

Furthermore, said multi-channel PMT allows to considerably decrease the noise to a non null mean, known as "dark current", outputted from every channel with respect to that of normal PMTs.

In a preferred embodiment of the device according to the present invention the dark current results half of that of the apparatuses based on normal PMTs at the highest amplification.

Furthermore, the device according to the present invention advantageously allows to perform more elaborate analyses, in virtue of the use of multiple intracellular and membrane fluorochromes at the same time, as only one multi-channel PMT can perform analyses on at least sixteen different transduction channels centered on sixteen different wavelengths.

An entirely digital conditioning chain, according to the present invention, includes the following stages: a. a procedure, designated baseline restoration, adapted to perform a mean in time such as to allow a compensation of the so-called dark current, of the thermal noise, of the so-called shot noise and of the laser oscillations; b. an amplification procedure adapted to perform the amplification of the signals by means of a modulation, appropriately varying the anodic gain of the optical system and improving the sensitivity and resolution; c. a procedure adapted to compensate the overlapping of particularly extensive spectral bands of photoluminescence signals of the fluorophores; d. a procedure adapted to perform a logarithmic amplification for a better representation of the signal dynamics; e. a procedure adapted to perform a Peak Detector stage which, through derivative controllers and through the selection of a threshold, allows to detect the peaks present in the signals acquired in a given time interval; f. a procedure adapted to perform the trigger stage for the generation of the start

scan for each signal related to each acquired channel; g. a procedure adapted to allow the switch for the selection of one or more trigger signals selectable among those available for a better filtering, h. a procedure adapted to perform a statistical processing of the signals and for the computation of the statistical parameters useful in cytofluorimetry, such as the variation coefficient and others; i. a procedure adapted to allow at least one diagrammatic representation of the signals on histograms and dot plots.

Some signal conditioning and processing stages are now disclosed in detail with reference to the chain diagram just shown.

A variant that implies an implementation by means of dedicated hardware is suggested in particular for the trigger stage, whereas the following stages suggested are performed by means of routine software instead of electronic circuitry, for which the optimal values of some numerical values, and the corresponding methods to obtain them, are indicated, thus solving the problem of the above mentioned false negatives. 1 ) Triggering stage

In a preferred embodiment of the apparatus the trigger stage is performed by means of a programmable PIC microcontroller which, according to the present invention, performs the signal conditioning in the digital domain.

Specifically a PIC 16f628 is preferred. A programming example of the internal memory of the microcontroller through a pic basic code is disclosed hereinafter: define osc 20 include "MODEDEFS.BAS" trisa=%1 1 1 11 trisb=%00000001 trigger var portb.O reset var portb.1 inputs var portb.2 start var portb.4 inputs=1 start=O

reset=0 pause 1 reset=1 main: if trigger=1 then goto Stabiliz goto main

Stabiliz: pauseus 1

Stand-by: if trigger=O then inputs=0 pauseus 1 start=1 pauseus 2 start=O pauseus 1 reset=0 pauseus 1 reset=1 inputs=1 else goto stand-by endif goto main end

Advantageously, the performing rate is higher with respect to the traditional electronic circuitry and the noise introduced results lower because of the subsequent reduction of the electronic circuitry.

In a preferred embodiment, to further increase the performance, said PIC and all of the related electronic configuration network are computer emulated or replaced with a routine software, integrally eliminating the electronic circuitry corresponding to the stage.

2) Method for the conversion of scales and for signal representation In order to replace the logarithmic amplification stage or the eeprom that serves as a look up table, a method has been suggested, which is especially suitable for cytofluorimetry that may readily be applied in a software routine adapted to perform a conversion from a linear scale to a logarithmic scale.

In cytofluorimetry, the fluorescence signals, which are translated into current signals and then into voltage by a current-voltage converter, may cover a range from a few μV to 10V. The method disclosed here, allows to obtain some numerical coefficients which allow to pass from one scale to another and vice versa.

Specifically, said method is displayed to pass from the 16 bit quantization to an 8 bit quantization, even though it may be applied to any conversion: a) a lower and a higher limit is set for each decade of one of the two representation systems involved in the conversion, e.g. 16 bits: i.e. the lowest output signal level from the optoelectronic system is assigned to channel 0, for instance 100 nV, so that channel 7 corresponds to 1 mV, channel 66 corresponds to 10 mV, channel 656 corresponds to 100 mV, channel 6554 corresponds to 1 V and finally channel 65535 corresponds to the maximum value, for instance to 10 V, outputted from the optical system; b) the previous a) is repeated with the other representation, e.g. 8 bits, involved in the conversion i.e. the numerical representations are defined after the logarithmic conversion in the 8 bit encoding system, specifically channel 0 corresponds to 100 nV, channel 55 to 1 mV, channel 103 to 10 mV, channel 154 to 100 mV, channel 205 to 1 V and channel 255 to 10 V; c) a rescaling of said signal outputted from the optical system is performed: specifically, a one-to-one correspondence is defined between the voltage values of said signal before and after the conversion with the lowest signal value outputted from the optical system which is referred to 0 V, whereas the highest value remains unvaried, scaling the intermediate values: e.g. it is set that 100 nV correspond to 0 V, 1 mV to 2 V, 10 mV to 4 V, 100 mV to 6 V, 1 V to 8 V and 10 V to 10 V; d) the limit values of each decade both in the linear system and in a logarithmic

scale are sorted in tables, which are similar to the tables in Figures 3 and 4. e) an interpolation is performed, which is drawn in the diagram in Figure 5, of the values in the table related to the logarithmic scale, thus obtaining a mathematical relation that expresses the correspondence between the two sets:

Y = a - b ^* In ( X + c ),

where y is the value after the conversion, x is the value before the conversion, a, b, c are constants computed by means of a numerical computation. For the specific application in the filed of cytofluorimetry the coefficient "b" may vary between 0 and -1. A preferred value has been computed to be -0.86859 and corresponds to the gain which varies as a function of the logarithmic base, and is expressed as a consequence of the doubling of the input; the coefficient "a" corresponds to the initial offset, which is totally independent from the input and may vary from 5 to 15, although the value of 8 is preferred; finally the coefficient "c" is a constant that may correspond to values in the range between 0 and 0.1 , although a value of 1.3017E-18 is preferred.

For the specific application in the filed of cytofluorimetry the coefficient "b" may vary between 0 and -1. A preferred value has been computed to be -0.86859 and corresponds to the gain which varies as a function of the logarithmic base, and is expressed as a consequence of the doubling of the input; the coefficient "a" corresponds to the initial offset, which is totally independent from the input and may vary from 5 to 15, although the value of 8 is preferred; finally the coefficient "c" is a constant that may correspond to values in the range between 0 and 0.1 , although a value of 1.3017E-18 is preferred.

The advantage resulting from the application of this method consists in being able to convert without any delay any channel value for a conversion factor in the range from 22 to 27, although 25.6 is preferred. For instance, if one should want to know which is the channel corresponding to a value of 2 V, it is sufficient to multiply it by 25.6.

It should be noted that said relation to perform the conversion may be personalized by varying the above said three parameters in order to obtain a

better resolution.

A further advantage consists in that such a procedure may also be applied for 20 bit representations, especially in consideration of the fact that only few cytofluori metric apparatuses offered by the known art reach at most 16 bit representations for the portion of conditioning chain that operates digitally.

Therefore, exploiting said method in an implementation of the apparatus according to the present invention, the use of a first analog-digital conversion stage is made possible with converters, one for each 20 bit channel so as to further reduce the quantization error. 3) Compensation stage of the overlapping of the signals

The compensation stage provides for the reduction or elimination of the overlaps produced by the various fluorescence signals, which as known occupy a very broad band, by means of routine software. Finally, said routine is able to eliminate the autofluorescence of the cells in a sample containing no fluorophores according to the following method:

- a first analysis is carried out on a "blank" sample, i.e. a sample having no markers, so as to store the autofluorescence data in a temporary file.

- a multi-parametric analysis is carried out on a new sample with markers, in which there are 15 markers or more, with the emission of different wavelengths; - said temporary file is retrieved by subtracting the signal levels computed in the previous step from those computed at the first step;

- a compensation of the signal overlaps is carried out through any matrix calculation technique allowing to define the percentages of overlapped signal to be subtracted from the previous step. The advantages of such an apparatus for cytofluorimetric analyses are apparent, in particular the use of a multi-channel PMT together with an optical fiber and calibrated interferential filters has allowed a substantial reduction of size and costs. Furthermore, it has been possible to replace every signal conditioning and processing stage with routine software, thus obtaining an entirely digital cytofluorimeter that allows analyses which have not been possible up until now due to the considerable background noise introduced by the electronic circuitry.

The present invention may be advantageously carried out by means of a computer software that includes encoding means for performing one or more steps of the method, when this program runs on a computer. Therefore, the protective scope is intended to extend to said computer software and also to computer readable means including a recorded message, said computer readable means including software encoding means for performing one or more steps of the method, when said software runs on a computer.

Variations may be made to the non-limitative example disclosed, without however departing from the protective scope of the present invention, including all of the equivalent embodiments for a person skilled in the art.

The specific modes to embody the invention described here do not limit the content of this application, which includes all of the variants of the invention according to the claims.