BACKGROUND TO THE INVENTION The differential count of white blood cells and leukocytes is widely used to assess the hematological and immunological status of human patients. Presently, about five manufactures supply the hematological analyzers used by the pathologists throughout the world which provide physicians with hematological biological results. These analyzers are, with different technologies, able to process about 100 to 150 patient samples per hour, and provide up 15 to 18 results per samples. They are able to analyze red blood cells, platelets and specific parameters relating thereto (Hemoglobin content, volume, distribution etc. ), and they are also able to analyze leucocytes circulating in the blood. For the last ten years different technologies have been used by the manufacturers to perform the white blood cell differential count which allows the enumeration of five different cell populations namely the polymorphonuclears (PMN) the polynuclears, eosinophils and basophils, the monocytes and the lymphocytes. These cells are normally resident in the blood, and are matured (or near mature) cells. They are derived from precursors located in the bone marrow. When an individual has a pathologic condition these precursors, or their neoplastic counterparts sometimes appear in the blood. Current analyzers are able to recognize them in a crude manner, but are not able to clearly quantify them. Present analyzers rely on expression flag (s) for quantification.

The expression of one or more flags allows the pathologists or his technologists to analyze the morphology of the cells under a microscope, and to perform manually a short differential count. The number cells per sample is limited to roughly 100. Should a cell count be especially low, it would require the analysis of several samples to obtain a statistically

meaningful cell count. Thus, the microscopic analysis method is time consuming (from 10 to 20 minutes per slide when the leukocyte count is low) and very inefficient.

For the last decade, flow cytometry (FCM) has emerged as an important tool for the diagnosis in haemato-oncology and in the follow up of immuno-deficient diseases. A common strategy for identifying blood components involves the use of monoclonal antibodies (Mabs) which recognise specific cell markers. Said Mabs are labelled with different fluorophores ; the capacity of the flow cytometers means that three and sometimes four different fluorescent signal can be recognised. Due to improvements in the measuring cell and an increase in the complexity thereof, a flow cytometer can measure 6 independent parameters in a sample.

These parameters are namely two physical parameters-side and forward scatters-and four fluorescence parameters, FL1 to FL4. A FCM device might be set up such that FL1 is sensitive to emission wavelength of fluorescein (FITC blue green light), FL2 is sensitive to phycoerythrin (PE, orange), FL3 to pteridine, cyanine or derivatives and/or tandems thereof (Per-CP, Per-CP Cy 5.5, PC5, far red), and FL4 to cyanin (APC, blue).

Such an approach is well known for classification of blood components. For example, FCM can be used to classify the blood lymphocytes into B, T and NK lymphocytes ; to subdivide T cells into helpers (marker CD4 positive) and cytotoxic/suppresors (marker CD8 positive); to subdivide T cells according to their activation level using HLA-DR expression. These determinations are routine work in many immunology laboratories and the same approach is now mandatory in hematopoietic pathology to classify lymphom, myeloma and leukemic cells.

To perform four color FCM analyses, one way is to combine four Mabs stained each with a different fluorophore suitable for the four FCM detectors. Another way is to use a method in which two Mabs are labelled with the same fluorophore ; this is the subject of a patent concerning detection of CD4, CD8, T and B cells. This means that two different cell types will be merged in the same fluorescence expression. The patent discloses that a distinction is made between same-labelled populations due to the expression of another discriminant marker. However, this method requires that a differentiating expression marker is available.

Blood comprises more clinically relevant cell types than the so-called"five population"cells detected by present hematology analyzers. Precisely enumerating lymphocytes in T, B and NK cells is of great importance because B cell proliferations are the most frequent in lympho- proliferative diseases. Quantitating CD8/HLA-DR positive cells allow the detection of virocytes (equivalent to the"atypical lymphocytes"flags of the analyzers) and are clinically relevant during AIDS follow up. Detecting bone marrow released precursors is important during inflammation/sepsis and myeloproliferative diseases or leukemias.

FCMs and/or devices attached thereto may, in a particular mode of operation, produce a two- dimensional intensity representation of data measured from a sample. The two-dimensional representation may be marked by the user into a certain number of regions to establish populations of cells ; the number of regions the device is able to compute is proportional to the number of populations that can be quantified. Because of the computation complexity required, the number of regions a FCM and/or device attached thereto can compute is limited, therefore, a limit is automatically placed on the number of populations a FCM can quantify.

US Patent number 5,843, 689 discloses a method for determining the immunoregulatory status of the mononuclear leukocyte immune system using FCM. The method uses light scatter determine the leukocyte class and fluorcescence data to enumerate the cell subclass and amount of activation antigen expression. The problem of measuring a large number of populations of components in the mononuclear leukocyte immune system is not encountered in the document and, therefore, is not addressed therein.

US Patent number 5,188, 935 discloses a method for lysing and fixing cells for use in FCM; the method discloses specific reagent-based methodological procedures, however, the problem of enumerating a large number of populations in a sample is not touched on.

US Patent number 5,627, 040 discloses a method for autoclustering of two-dimensional arrays derived from FCM. There is no disclosure therein of enumerating a large number of populations in a sample.

There is a need for a method of easily analyzing the populations of biological entities in a sample which is capable of simultaneously measuring the size of the populations of at least two, and ideally more than five biological entities, such as blood components. There is a need for overcoming the limits placed on FCM devices, wherein the number of populations to be measured is greater than the number of different fluorescent markers that can be simultaneously detected. There is a need for overcoming the limits placed on FCM devices, wherein the number of populations to be measured is limited by the number of regions into which a two-dimensional intensity representation of the data therefrom can be demarked.

SUMMARY OF THE INVENTION One embodiment of the present invention is a method suitable for quantifying a sample comprising populations of biological entities, using a flow cytometer capable of measuring forward light, scattered light and fluorescence comprising: a) labelling populations of interest, optionally wherein two or more populations are labelled by the same fluorophore, b) taking a flow cytometry measurement of said sample, c) demarking one or more cluster regions, d) performing one or more Boolean operations upon said cluster regions, and e) obtaining quantitative data relating to the size of said populations.

Another embodiment of the present invention is a method as described above wherein two or more said regions overlap.

Another embodiment of the present invention is a method as described above wherein step a) is performed using population-specific antibodies.

Another embodiment of the present invention is a method as described above wherein labels of step a) are one or more of fluoresceine, phycoerythrin, pteridine, cyanine, Per-CP, Per- CP, Cy 5.5, PC5, cyanin, APC.

Another embodiment of the present invention is a method as described above wherein said Boolean operations are performed according to the scheme: Population Boolean operations Polymorphonuclears R1'and R4'and R6' Immature myeloids R1'and R5'and R6'and not R12' Monocytes R1'and R3'and R8'and not R9' Eosinophiles R1'and R4'and R5'and R12' Basophiles R2'and R12'and R15'and R10'and not R9' Lymphocytes R1'and R2'and not R15' T lymphocytes R1'and R2'and R4' B lymphocytes R1'and R2'and R10'and (R9'and not R7') CD8+ T Lympho R1'and R2'and R4'and R7'and R9' CD8+ DR+ virocytes R1'and R2'and R4'and R7'and R9'and R10' Non CD8+ T lympho and R2'and R7'and not R9' non T non B lympho and R2'and not (R4'or R9'or R15') Nucleated Red Blood Cells R16'and not R10' Plasmatocytes R15'and R13'and R11'and not R13' Blasts R15'and R13'and not (R11'or R7') Events R1' Another embodiment of the present invention is a method as described above wherein said Boolean operations are performed according to the scheme : Population Boolean operations Polymorphonuclears (R2 and R5 and R7) and not (R4 or R6 or (R1 and R14 and R15) or Poiymorphonuciears (R2 and R5 and R7) and not (R4 or R6 or (R1 and R14 and R15) or R11 or R12 or R13) immature myeloids (R2 and R6 and R7) and not (R3 or R4 or R10 or R11 or R13 or R14) Monocytes (R1 and R14 and R15) and not (R3 or ( (R5 and not R4) and R2) or (R4 and R5)) Eosinophiles ( (RI and R3) and R5 and R6 and R9) and not (R4 or R10 or R11 or R12 or R13 or R14) Basophiles (R1 and R3 and R4 and R6 and R10) and not (R5 or R11 or R12 or R13 or R14 or R15) Lymphocytes (R1 and (R13 or (R4 and R5)) or (R9 and R10) or R8) and not (R1 or R14 or R15) T lymphocytes (RI and R4 and R5 and R13) and not ( (RI and R14 and R15) or (R10 and R11) or (R9 and not R10)) B lymphocytes (R1 and R10 and R11) and not ( (R1 and R14 and R15) or (R2 and R7 and (R5 or R6) ) or (R1 and R3 and R4 and R6 and R10) or (R1 and R4 and R16)) Non T nonB lympho (R1 and R4 and R5) and not (R13 or (R1 and R14 and R15) or (R1 and R3 and R4 and R6 and R10) or (R1 and R4 and R16) or (R10 and R11)) CD4+ T Lympho (R1 and R8 and R9 and R10 and R13) and not (R7 or (R1 and R4 and R16) or (R1 and R3 and R4 and R6 and R10) or (R1 and R14 and R15) or (R10 and R11)) Non CD4+ T lympho (R1 and R7 and R8 and R13) and not (R9 or (R1 and R4 and R16) or (R1 and R3 and R4 and R6 and R10) or (R1 and R14 and R15) or (R10 and R11)) CD4+ DR+ T lympho (R1 and R4 and R5 and R8 and R9 and R11 and R13) and not (R10 or (R1 and R14 and R15) or (R1 and R4 and R16)) Atypical lymphocytes (R1 and R4 and R5 and R7 and R8 and R11 and R13) and not (R10 or (R1 and R14 and R15) or (R1 and R4 and R16)) Nucleated Red Blood (R1 and R4 and R16) and not ( (R1 and R3 and R4 and R6 and R10) Cells or R8 or R10 or R11 or 12) Blastes (R1 and R12) and not ( (R1 and R14 and R15) or ( (R5 or R6) and not R4) or R10 or (R1 and R4 and R16)) Events (R1 and ( (RI 4 or R15) or R13 or (R4 and R5) ) or (R10 and R11) or (R4 and R16) or R12) ) or (R2 and ( (R5 or R6) and not R4) ) or (R3 and R6 and R9)

Another embodiment of the present invention is a computer program, stored on a computer readable medium, capable of performing a method as described above.

Another embodiment of the present invention is a computer storage medium comprising a computer program as described above.

AIMS OF THE PRESENT INVENTION One aim of the present invention is to provide a method suitable for quantifying a sample comprising populations of biological entities, using a flow cytometer capable of measuring forward light, scattered light and fluorescence comprising: a) labelling populations of interest, optionally wherein two or more populations are labelled by the same fluorophore, b) taking a flow cytometry measurement of said sample, c) demarking a number of cluster regions, d) performing one or more Boolean (gating) operations upon said cluster regions, and e) obtaining quantitative data relating to the size of said populations.

By labelling two or more populations with the same fluorophore, and using Boolean operations to distinguish between said populations, the number of populations that can be detected is maximised. This is especially relevant when the number of fluorophores available for detection is less than the number of populations to be detected. This scenario occurs in the analysis of blood wherein the sizes of the populations of 6 or more blood components need to be measured, and flow cytometry devices may detect only 3 or 4 fluorescent labels simultaneously.

Another aim of the present invention is to provide said method, using non-covalent association to label populations of interest. Using a non-covalent means of labelling a population which takes advantage of a unique aspect of the surface of the population, provides a non-invasive, sensitive and simple means for identifying entities using FCM according to the invention.

Another aim of the present invention is to provide said method, using demarcation of regions on two dimensional intensity plots wherein said two or more said regions overlap. The demarcation of two dimensional intensity plots into overlapping regions allows the user artificially to overcome the limits set by FCMs and/or devices attached thereto regarding the maximum number of regions available for Boolean operations; this subsequently allows more components in a sample to be simultaneously analysed.

Another aim of the invention is to provide said method, making use of one or more sets of Boolean operations that are performed between demarked regions of two dimensional intensity plots. The use of Boolean logic, in combination with a computing device, allows operations to be performed extremely quickly since Boolean logic is a fundamental operation of computers.

Another embodiment of the present invention is a computer program, stored on a computing device having a data storage means, which is capable of performing a method according to the invention. The use of a computer program to perform certain steps of the invention allows the method to be transferred to any FCM device which comprises a computer and/or is capable of interfacing with a computer, so representing a considerable time saving, cost saving and convenience in performing the method of the invention.

DETAILED DESCRIPTION OF THE PRESENT INVENTION One embodiment of the present invention is a method for quantifying a sample comprising populations of biological entitles, using a flow cytometer capable of measuring forward light, scattered light and fluorescence comprising: a) labelling populations of interest, optionally wherein two or more populations are labelled by the same fluorophore, b) taking a flow cytometry measurement of said sample, c) demarking a number (C) of cluster regions, d) performing one or more Boolean (gating) operations upon said cluster regions, and e) obtaining quantitative data relating to the size of said populations.

By population herein means a collection of one or more of the same entity.

By entity herein means any biological molecule detectable using a fluorescent label and a FCM. Examples of entities include, but are not limited to any blood component, any whole blood component, any immune system-related blood component, blood leukocytes, lymphocytes, platelets, polymorphonuclears, immature myeloids, eosinophils, basophils, T lymphocytes, non T non B lymphocytes, CD4+ T lymphocytes, Non CD4+ T lymphocytes, CD4+ DR+ T lymphocytes, CD8+ T lymphocytes, Non CD8+ T lymphocytes, CD8+ DR+ T lymphocytes, atypical lymphocytes, nucleated red blood cells, blasts. Entities include any cell from any organism, including, but limited to eukaryotic cell, prokaryotic cell, viruses, fungi,

plant cells. Entities include any biological macromolecule, including, but not limited to proteins, nucleic acids, glycoproteins, protein-nucleic acid associations, bacteria, protozoa, protoplasts, zooplankton etc.

The term"flow cytometer"can be readily understood by a skilled person and encompasses flow cytometers of any kind, as well as any analyzers, in particular hematological analyzers, which employ the same or similar technological platform.

According to the invention, a number (P) of populations can be labelled by the same fluorophore. The number of populations labeled by the same fluorophore can be two or more.

It may be, for example, 2,3, 4,5, 6,7, 8,9, 10,10 or more. The value of P can depend of many factors including the number of regions the cytometer or device attached thereto is capable of performing Boolean operations thereon, and the number of different fluorophores said cytometer is capable of simultaneously detecting.

In another embodiment of the present invention, the labelling of populations of interest may be performed by any means known in the art. It may, for example, be performed by using a protein that interacts with a macromolecule on the surface of the population of entities, said protein being tagged with a fluorophore. It may be performed using an antibody directed against an antigen belonging to a population of entities; said antibody being tagged with a fluorophore. Using a non-covalent means of labelling a population which takes advantage of a unique aspect of the surface of the population, provides a non-invasive, sensitive and simple means for identifying entities using flow cytometry according to the invention. When monoclonal antibodies are used labeled with the same fluorphore, it is an aspect of the invention that they do not recognise antigens common to different populations of cells.

Preferably, one fluorescence channel dedicated to the fluorescent tag of an antibody separating clearly the lymphoid cells, the myeloid cells and the blast cells. This role is generally devoted to the marker CD45. in association with the physical parameters (i. e. side scatter and forward scatter).

According to one aspect of the present invention, the flow cytometer measures side scatter, forward scatter, and the fluorescence of one or more fluorescent labels. The data may be

presented as two dimensional intensity plots wherein the two axis are any combination of the aforementioned measurements. For example, side scatter vs. forward scatter, side scatter vs. fluorescence due to chromophore 1, fluorescence due to chromophore 1 vs. fluorescence due to chromophore 2, etc. Providing two dimensional data plots of combinations of the data available allows the cluster regions to be readily identified and allows Boolean operations to be performed thereon quickly. The use of two dimensional analysis allows the method of the invention to be performed on the basic flow cytometers, and does not require extensive multidimensional array handling capabilities.

Alternatively, according to another aspect of the present invention, the data may be processed as a multidimensional intensity plot wherein the axes are any combination of the aforementioned measurements. Manipulating multidimensional data allows fast and integrated calculations to proceed, without the need to analyse separate two dimensional data. It further allows easy compression of data, so saving storage space, time and expenditure on storage.

According to another aspect of the present invention, regions are identified in the two-or multi-dimensional representations of the aforementioned parameters. The regions are delimited according to the clustering of data points. Identification of regions is performed by methods of the art, and may be automated and/or manual.

The number of regions that may be indicated and made available for Boolean (gating) operations can be any number. Alternatively, it may be limited by the flow cytometer and/or the processing device attached thereto; the mathematical complexity and the size of the data sets sometimes means the number or regions a device can perform Boolean operations on is set at a limit. Often this limit is below that required to quantify populations of entities in a sample. For example, if a user has to identify 15 components of blood, 30 regions would need to be defined and operated on in a gating strategy. This number is beyond the level set by some devices. Therefore, the inventors have provided another aspect of the invention which is to demark two dimensional intensity plots into bi-lobed regions and overlapping regions to increase artificially the number of regions. When used according to the invention, bi-lobed regions and overlapping regions overcome the limits placed on the number of region numbers by some flow cytometers.

Another aspect of the invention is the demarcation of regions on a two dimensional intensity plot, wherein the two axis are any combination of the aforementioned measurements, and two or more regions overlap.

The regions may be of any shape including, but not limited to circular, trapezoidal, concave, polygonal, and bi-lobed, tri-lobed, multi-lobed etc.

According to one aspect of the invention, the demarcation is performed using any method of the art. According to another aspect of the invention, the demarcation is performed using a scheme depicted in Figure 1. According to another aspect of the invention, the demarcation is performed using a scheme depicted in Figure 2-1 and 2-2. According to another aspect of the invention, the demarcation is performed using a set of rules described below. The demarcation of two dimensional intensity plots into overlapping regions allows a user artificially to overcome the limits set by flow cytometers and/or devices attached thereto regarding the maximum number of regions available for gating operations; this subsequently allows more components in a sample to be simultaneously analysed.

According to one aspect of the invention, the rules to design Regions, Gates and the Boolean take in account the following : To define a cluster (literally a cell population), at least two regions defined in dot plots and three or more different parameters (fluorescence or physical) are frequently required.

Considering that, a minimum of fifteen clusters may be defined in the present invention, it requires defining a minimum of 24 regions. In the Cell Quest@ software of Becton Dickinson the number of available regions is 16. It is why in the present discovery the inventors have been obliged to create artificial new regions by combination of the available regions.

The combinations may take the form of: 1) Simple combination: two regions overlap creating three gates. "C = R1 AND R2"."A = R1 AND NOT R2""B = R2 AND NOT R1". The clusters are closed together, necessarily.

See Figure 4-1.

2) Simple combination with the use of bi-lobed region. Same principle as above but the clusters are not closed together, necessarily. See Figure 4-2.

3) Complex combination using three overlapping regions defining 5 gates. The five gates are defined as:"A = R1 AND NOT R3", "B = R3 AND NOT (R1 OR R2) ","C = R2 AND<BR> NOT R3", "D = R1 AND R3", "E = R2 AND R3". See Figure 4-3.

It is of course possible to use any combination of the above e. g. one or more bi-lobed region in the complex combination using three overlapping regions.

According to another aspect of the present invention, the gating strategy (Boolean operations) is performed between regions demarked. Said operations may be performed by methods known in the art. Alternatively, they may be performed according to a set of rules calculated according to a method of the invention. Alternatively, they may be performed according to Table 1. Population Boolean operations a Polymorphonuclears (R2 and R5 and R7) and not (R4 or R6 or (R1 and R14 and R15) or R11 or R12 or R13) Immature myeloids (R2 and R6 and R7) and not (R3 or R4 or R10 or R11 or R13 or R14) Monocytes (R1 and R14 and R15) and not (R3 or ( (R5 and not R4) and R2) or (R4 and R5)) Eosinophiles ((R1 and R3) and R5 and R6 and R9) and not (R4 or R10 or R11 or R12 or R13 or R14) Basophiles (R1 and R3 and R4 and R6 and R10) and not (R5 or R11 or R12 or R13 or R14 or R15) Lymphocytes (R1 and (R13 or (R4 and R5)) or (R9 and R10) or R8) and not (R1 or R14 or R15) T lymphocytes (R1 and R4 and R5 and R13) and not ( (RI and R14 and R15) or (R10 and R11) or (R9 and not R10)) B lymphocytes (R1 and R10 and R11) and not ( (R1 and R14 and R15) or (R2 and R7 and (R5 or R6)) or (R1 and R3 and R4 and R6 and R10) or (R1 and R4 and R16)) Non T nonB lympho (R1 and R4 and R5) and not (R13 or (R1 and R14 and R15) or (R1 and R3 and R4 and R6 and R10) or (R1 and R4 and R16) or (R10 and R11)) CD4+ T Lympho (R1 and R8 and R9 and R10 and R13) and not (R7 or (R1 and R4 and R16) or (R1 and R3 and R4 and R6 and R10) or (R1 and R14 and R15) or (R10 and R11)) Non CD4+ T lympho (R1 and R7 and R8 and R13) and not (R9 or (R1 and R4 and R16) or (R1 and R3 and R4 and R6 and R10) or (R1 and R14 and R15) or (R10 and R11)) CD4+ DR+ T lympho (R1 and R4 and R5 and R8 and R9 and R11 and R13) and not (R10 or (R1 and R14 and R15) or (R1 and R4 and R16)) Atypical lymphocytes (R1 and R4 and R5 and R7 and R8 and R11 and R13) and not (R10 or (R1 and R14 and R15) or (R1 and R4 and R16)) Nucleated Red Blood (R1 and R4 and R16) and not ( (R1 and R3 and R4 and R6 and R10) Cells or R8 or R10 or R11 or 12) Blastes (R1 and R12) and not ( (R1 and R14 and R15) or ( (R5 or R6) and not R4) or R10 or (R1 and R4 and R16)) Events (R1 and ( (R14 or R15) or R13 or (R4 and R5) ) or (R10 and R11) or (R4 and R16) or R12) ) or (R2 and ( (R5 or R6) and not R4) ) or (R3 and R6 and R9)

TABLE 1: Boolean operations involving regions demarked from two dimensional intensity representations of FCM data, and the population type derived from said operations.

According to the strategies in Table 1, R3 is bi-lobed, surrounding the eosinophiles and the basophiles. R5 overlaps R4 and R6 (surrounding lymphocytes and eosinophiles) ; R6 overlaps R4 surrounding the non T lymphocytes. R8 is bi-lobed surrounding CD4 and non CD4 T lymphocytes. R7 overlapped the left side of R8. R9 overlapped R10 to surround the CD4 T cells.

Exemplary demarcations of regions (R1 to R16) for use in the strategy (or algorithm) of Table 1 are illustrated in Figure 1-1 and 1-2 (A to H) and explained below. On an SSC (side scatter; y-axis) /FSC (forward scatter; x-axis) dot plot in Figure 1-1A, region 1 (R1) surrounds mononuclear cells including the blasts area. Region 2 (R2) surrounds the myeloid cells (PMN and immature granulocytes). The overlap between R1 and R2 is not intended to create a new

region but is due to the necessity to increase the surface of the regions to contain the inter individual variability. R3 is a bi-lobed region surrounding the basophiles (low SSC) and the eosinophiles (high SSC). On a CD16-CD2-CD71 FITC (y-axis) /SSC (x-axis) dot plot in Figure 1-1B, R4 is approximately rectangular on the left and surround lymphocytes and NRBC, R6 is oblique and rectangular in the bottom and surrounds eosinophiles (combination of R5 and R6), Immature granulocytes (R6 and not (R5 or R4) ) and non T lymphocytes (R6 and R4). R5 is rectangular with two extensions, one in the left in the middle of R4 surround the T lymphocytes (R4 and R5) and one in the bottom right overlapping with R6 creating a new region surrounding eosinophiles (R5 and R6). R5 surrounds mature polymorphonuclear (R5 and not (R6 or R4) ). On a CD14-CD34-CD5 APC (y-axis) /CD19-CD123-CD4 PE (x- axis) dot plot R7 in Figure 1-1C is an"L"-shape and R8 is bi-lobed region, with the left lobe of R8 overlapping with R7 detremining a new region surrounding non CD4 T cells. The right lobe surrounding the CD4 T cells. On CD16-CD2-CD71 FITC (y-axis) /CD19-CD123-CD4 PE (x-axis) dot plot in Figure 1-1D R9 is a"dog leg right"and R10 is trapezoidal ; together they overlap and surround the CD4+ region as indicated with an arrow. R9 is designed to improve eosinophiles definition and R10 the B cells definition. The remaining regions are depicted in Figure 1-1 E where R11 surrounds the HLA-DR positive cells (blastes, B cells monocytes and virocytes), Figure 1-1 F where R12 is to increase the definition of blastes and Figure 1-2G and H with y-and x-axis labeled to identify the measured signals and R13 improving the T cell definition, R16 and R14 the monocytes definition, and R16 the NRBCs.

Alternatively, in another embodiment, the gating strategy (Boolean operations) may be performed according to Table 2. Population Boolean operations Polymorphonuclears R1'and R4'and R6' Immature myeloids R1'and R5'and R6'and not R12' Monocytes R1'and R3'and R8'and not R9' Eosinophiles R1'and R4'and R5'and R12' Basophiles R2'and R12'and R15'and R10'and not R9' Lymphocytes R1'and R2'and not R15' T lymphocytes R1'and R2'and R4' B lymphocytes and R2'and R10'and (R9'and not R7') CD8+ T Lympho R1'and R2'and R4'and R7'and R9' CD8+ DR+ virocytes R1'and R2'and R4'and R7'and R9'and R10' Non CD8+ T lympho and R2'and R7'and not R9' non T non B lympho and R2'and not (R4'or R9'or R15') Nucleated Red Blood Cells R16'and not R10' Plasmatocytes R15'and R13'and R11'and not R13' Blasts R15'and R13'and not (R11'or R7') Events R1'

TABLE 2: Boolean operations involving regions demarked from two dimensional intensity representations of FCM data, and the population type derived from said operations.

Exemplary demarcations of regions (R1'to R16') for use in the strategy (or algorithm) of Table 2 are illustrated in Figure 2-1 (A to G) and explained below. On CD45 Per-CP (y-axis)/ CD16b-CD71-CD3 FITC (x-axis) dot plot in Figure 2-1A Region 1' (R1') surrounds approximately all the hematopoietic cells including the NRBC. As blasts cells could be CD45 dim or low, R1'do not surround the blasts. R1 represent 100% hematopoietic cells and is the basis of all the others cells population percentage. Region 16' (R16') is included in R1'and <BR> <BR> surrounds the NRBC (i. e. , CD71 positive CD45 low or negative) myeloid cells (PMN and immature granulocytes). The overlap between R1'and R16'is intended to include the percentage of NRBC in the differential count. On SSC (y-axis) /CD45 Per-CP dot plot in Figure 2-1 B, each region surrounds a big family of cells, which is common in pivotal dot plots such as this one. R2'surrounds the lymphocytes, R3'the monocytes, R6'the myeloid family, and R15'the CD45 low represented by blasts, plamatocytes and basophiles. R2'and R15' overlap to create a new region which surrounds the basophile area. On CD16b-CD71-CD3 FITC (y-axis) /SSC (x-axis) dot plot on Figure 2-1 C R4'covers all FITC positive cells (i. e. , in the SSC low area the CD3 positive T cells and in the SSC medium or high the CD16b mature polymorphonuclears) and R5'surrounds more immature myeloid cells (from band cells to myeloblasts). R4'and R5'overlap to create a new region surrounding the eosinophiles CD16b intermediate and high SSC. On CD16b-CD71-CD3 FITC (y-axis)/CD19-CD8 APC (x- axis) dot plot in Figure 2-1 D different cell populations are labeled by the same fluorophore.

Namely, CD8 positive cells and B cells are stained with FITC and distinguished by CD3 staining, since CD8 positive cells belonging to the T cells family are also CD3 positive while B

cells are CD3 negative. CD8 positive cells are surrounded by a new region created by the overlap of R7'and R9'. The B cells (FITC positive but negative for CD16b, CD71 and CD3) are surrounded by the lower part of R9' (R9'and not R7'). In CD38-HLA-DR-CD123 PE (y- axis)/CD16b-CD71-CD3 FITC (x-axis) dot plot in Figure 2-1 E there is one region, R10', with an L shape. In the FITC negative part of the plot the limit separates the HLA-DR positive non T lymphocytes cells (CD3 FITC negative). These HLA-DR positive CD3 negative cells belong to the B cells population. The NK cells are indirectly determined by this gating because they are CD3 (and CD16b which is a strictly polymorphonuclear marker in contrast to CD16 and CD16a which are mainly expressed by NK cells) negative and HLA-DR negative. In this same part of the region the basophile are located because they are CD123 positive (the separation of the basophiles PE stained by CD123 and B lymphocytes PE stained by HLA-DR is made using differential expression of CD45; an overlap might occur between basophiles and blasts and basophiles and CLL B cells, such cells having frequently a low CD45 expression). The right part (FITC positive) of the R10'region is dedicated to the T cells positively stained for HLA-DR, and particularly the CD8 positive T cells, useful marker of viral infection (activated T cytotoxic cells, virocytes). In CD38-HLA-DR-CD123 PE (y-axis) /CD8-CD19 APC (x-axis) dot plot in Figure 2-1 F R8'is a redundancy of R10'and R11'surrounds the plasmatocytes region (CD19 intermediate and CD38 extremely bright). In SSC (y-axis) /FSC (x-axis) dot plot in Figure 2-1 G the classical physical cells parameters (size and complexity) are used to better define eosinophiles (SSC high and FSC intermediate) and basophiles FSC low and SSC low (inside the lymphocytes area). The bi-lobed shape of the R12'region serves to create artificially two regions because basophiles and eosinophiles are different in their phenotypes and it is easy to split R12'using Boolean logic. Hence, R12'is bi-lobed and surrounds the eosinophiles and the basophiles. R13'is dedicated to surround blasts and plasmatocytes. It is not a mononuclear cells gate. Blasts location is generally under the monocytes (lower SSC) and partially overlaps the lymphocyte region.

Figures 3-1 and 3-2 show exemplary gating using regions R1'to R16'as above in a sample from a patient with a Myeloproliferative syndrome in acutisation, showing the blasts location (arrows) in all the dot graphs and the immature granulocytes.

Another embodiment of the present invention is a device capable of performing the method of the present invention. In one aspect of the invention the device is a FCM capable of

performing the method of the present invention. In another aspect of the invention, the device interfaces with a FCM and enables the FCM to perform the method of the invention.

Another embodiment of the present invention is a device capable of performing steps c) and/or d) and/or e) according to the invention. In one aspect of the invention, the device may be connected directly to the FCM and/or it receives data from the FCM without direct connection to the FCM. Data may be received by any means known in the art, including, but not limited to, storage media such as floppy disk, optical disk (e. g. MO, CD, DVD), DAT; across a network; wireless communication; IR communication.

Another embodiment of the present invention is a computer program, stored on a computing device having a data storage means, which is capable of performing steps c) and/or d) and/or e) according to the invention. The computer program is written according to methods known in the art. According to one aspect of the invention, the computer program has a user interface which allows the operation of the FCM and/or the method of the invention; said user interface presenting options, displays, dialogue boxes in accordance to methods known in the art. According to another aspect of the invention, the computer program interacts with a computer program installed on the FCM, so allowing the method of the invention to be performed by the FCM computer program.

Another embodiment of the invention is a data media, such as a floppy disk, CD-ROM, DVD, comprising the computer program according to the invention.

FIGURES Figure 1-1 and Figure 1-2, (A) to (H), show various dot plots, wherein regions have been demarked. Regions R1 to R16 in the figures are exemplary for use in the strategy (or algorithm) as detailed in Table 1.

Figure 1-1 (A): FSC/SSC dot plot Region 1 surrounds mononuclear cells including the blasts area Region 2 surrounds the myeloid cells (PMN and Immature granulocytes). The overlap between Region 1 and Region 2 is not intended to create a new region but is due to the necessity to increase the surface of the regions to contain the inter individual variability.

Region 3 is a bi-lobed region surrounding the basophiles and the eosinophiles.

Figure 1-1 (B): CD16-CD2-CD71 FITC/SSC dot plot A complex situation with three regions is depicted. R4 is approximately rectangular on the left ; R6 is oblique and rectangular in the bottom and R5 is rectangular with two extensions, one in the left in the middle of R4 and one in the bottom right overlapping with R6. R4 and R5 surround the T lymphocytes and R6 and R5 surround eosinophiles.

Figure 1-1 (C): CD14-CD34-CD5/CD19-CD123-CD4 dot plot Shows two regions; R7 is an"L"-shape and R8 is bi-lobed. The left lobe of R8 overlaps R7.

Figure 1-1 (D): CD16-CD2-CD71/CD19-CD123-CD4 dot plot Shows two regions; R9 is a"dog leg right"and R10 is trapezoidal. They overlap surrounding the CD4 + region (indicated).

Figures 1-1 (E), (F), Figure 1-2 (G), (H) dot plot Depict plots of one or more single region.

Figure 2-1 and 2-2, (A to L), show various dot plots, wherein regions have been demarked.

Regions R1'to R16'in these figures are exemplary for use in the strategy (or algorithm) as detailed in Table 2.

Figure 2-1 (A): CD45/CD16b-CD71-CD3 FITC dot plot

Region 1' (R1') surrounds approximately all the hematopoietic cells including the NRBC. As blasts cells could be CD45 dim or low, R1'do not surround the blasts. R1'represent the 100% hematopoietic cells and is the basis of all the others cells population percentage.

Region 16' (R16') is included in R1'and surrounds the NRBC (CD71 positive CD45 low or negative) myeloid cells (PMN and Immature granulocytes). The overlap between R1'and R16'is intended to include the percentage of NRBC in the differential count.

Figure 2-1 (B): SSC/CD45 PerCP dot plot Depicts a pivotal dot plot and each region surrounds a big family of cells as is common using this plot. R2'surrounds the lymphocytes, R3'the monocytes, R6'the myeloid family, and R15'the CD45 low represented by blasts, plamatocytes and basophiles. R2'and R15' overlap to create a new region which surrounds the basophile area.

Figure 2-1 (C): CD16b-CD71-CD3 FITC/SSC dot plot Depicts two regions; R4'covers all FITC positive cells (i. e. , in the SSC low area the CD3 positive T cells and in the SSC medium or high the CD16b mature polymorphonuclears). R5' surrounds more immature myeloid cells (from band cells to myeloblasts) (better seen in Figure 3-1). R4'and R5'overlap to create a new region surrounding the eosinophiles CD16b intermediate and high SSC.

Figure 2-1 (D): CD16b-CD71-CD3 FITC/CD19-CD8 APC dot plot This plot exemplifies the situation wherein two or more populations are labeled by the same fluorophore. CD8 positive cells and B cells are stained with FITC and distinguished by another staining, here CD3. CD8 positive cells belonging to the T cells family are also CD3 positive. B cells are CD3 negative. In this plot CD8 positive cells are surrounded by a new region created by the overlap of R7'and R9'. The B cells (FITC positive but negative for CD16b, CD71 and CD3) are surrounded by the lower part of R9' (R9'and not R7').

Figure 2-1 (E): CD38-HLA-DR-CD123 PE/CD16b-CD71-CD3 FITC dot plot There is one region, R10', with an L shape. In the FITC negative part of the plot the limit separates the HLA-DR positive non T lymphocytes cells (CD3 FITC negative). These HLA- DR positive CD3 negative cells belong to the B cells population. The NK cells are indirectly determined by this gating because they are CD3 (and CD16b which is a strictly

polymorphonuclear marker in contrast to CD16 and CD16a which are mainly expressed by NK cells) negative and HLA-DR negative. In this same part of the region the basophile are located because they are CD123 positive (the separation of the basophiles PE stained by CD123 and B lymphocytes PE stained by HLA-DR is made using differential expression of CD45; an overlap might occur between basophiles and blasts and basophiles and CLL B cells, such cells having frequently a low CD45 expression). The right part (FITC positive) of the R10'region is dedicated to the T cells positively stained for HLA-DR, and particularly the CD8 positive T cells, useful marker of viral infection (activated T cytotoxic cells, virocytes).

Figure 2-1 (F): CD38-HLA-DR-CD123 PE/CD8-CD19 APC dot plot R8'is a redundancy of R10'and R11'surrounds the plasmatocytes region (CD19 intermediate and CD38 extremely bright).

Figure 2-1 (G): SSC/FSC dot plot The classical physical cells parameters (size and complexity) are used to better define eosinophiles (SSC high and FSC intermediate) and basophiles FSC low and SSC low (inside the lymphocytes area). The bi-lobed shape of the R12'region serves to create artificially two regions because basophiles and eosinophiles are different in their phenotypes and it is easy to split R12'using Boolean logic. R13'is dedicated to surround blasts and plasmatocytes. it is not a mononuclear cells gate. Blasts location is generally under the monocytes (lower SSC) and partially overlaps the lymphocyte region.

Figures 2-1 (H) (I) : CD45 PerCP/dot plot These dot plots are not for cell population definition but to attempt visual control of some interesting population separation. Plot H shows only the"lymphocytes CD8"gated events and is a control of the presence of CD8 T lymphocytes expressing HLA-DR (namely virocytes).

These cells are easily visually separated from CD8 positive HLA-DR negative cells. Plot I is a visual control of the lymphocytes populations (plot expressed events fitting the lymphocytes gate Boolean logic) CD8 positive T lymphocytes ; B cells ; CD8 negative T cells (indirectly the CD4) and non T non B cells (indirectly the NK cells).

Figure 2-2 (J) (K): These dot plots are also visual control in the classical FSC/SSC and CD45/SSC dot plot.

Figure 2-2 (L): It is a visual control of the events gated in the blasts gate. The leukemic blasts expressed great phenotype variability and the gating strategy is highly sensitive but relatively poorly specific as many unlysed red blood cells and debris might contaminate this gate. This plot gives the opportunity to see if the gated events are in cluster or invade the R13 region by the left as the debris do.

Figure 3-1 and 3-2 are an example of gating using the regions depicted and explained in Figure 2-1 and 2-2 and in relation to Table 2 in a patient with a Myeloproliferative syndrome in acutisation showing the blasts location (arrows) in all the dot graphs and the immature granulocytes.

Figure 4-1,4-2, and 4-3 schematically illustrates regions and gates. The regions R1, R2 and R3 are only schematic representations and are not the same as in Figures 1-1 and 1-2 and Table 1.

Figure 4-1 shows a combination of two regions R1 and R2 which overlap to create three gates A, B and C.

Figure 4-2 shows (i) a combination of one bi-lobed region (R2) and a single region R1 which overlap to create three gates A, B and C, and (ii) a combination of two bi-lobed regions (R1 and R2 which overlap to create three gates A, B and C.

Figure 4-3 shows (i) a combination of three regions (R1, R2, and R3) which overlap to create five gates A, B, C, D and E.