METHOD AND APPARATUS FOR SCREENING MICROSCOPIC CELLS IN AGED SAMPLES

Abstract: A method and apparatus (10, 40, 56, 148, 178, 210, 292) for automatically retrieving, counting and/or analyzing at least one selected white blood cell population and/or subset thereof of an aged whole blood sample or portion thereof. A volume of a biological sample (12, 42, 150, 180, 294) containing the white blood cells is prepared and at least one reactant (16, 46, 154, 184, 298) specific or preferential at least to some selected biological cells is introduced thereto and rapidly mixed. The total white blood cell populations and lymphocyte population are first electronically counted and then the neutrophil population including the aged neutrophil population is removed from the same or a second sample portion. The opacity and/or volume parameter of the cells then can be modified and the mixture then again counted and analyzed to obtain the desired white blood cell population analysis.

Description: METHOD AND APPARATUS FOR SCREENING MICROSCOPIC CELLS IN AGED SAMPLES Technical Field This invention relates generally to a method and apparatus for screening cells in aged samples. More particularly, the invention is directed to an analysis of the aged cells by utilizing microspheres having specific monoclonal antibodies bound thereto to remove one cell population and to obtain a multi-part white blood cell population differential. This invention relates generally to an automated analyzer and methods of using same for screening biological cells or formed bodies for the enumeration of populations which express selected chaiacteristics for research, diagnostic, medical or industrial purposes. More particularly, the automated analyzers and methods embodying the invention enable multiple part classifications of cells and formed bodies, functional phenotyping of cells and formed bodies, typing of leu emic, lymphoma and solid tumor cells, among others, using a unique combination of electronic and optical technology and the specificity of selective biological molecules, such as antibodies, for such screening and selective enumeration of the cells and formed bodies. Background Art Automation of routine complete blood cell (CBC) analysis of human peripheral blood by an automated blood cell counter was successfully achieved by the COULTER COUNTER (Registered Trademark) Model A of Coulter Electronics, Inc. of Hialeah, Florida. The electronic particle sensing system principle of that instrument is disclosed in U.S. Patent No. 2,656,508 issued October 20, 1953 to Wallace H. Coulter. The use of optical sensing means or lasers, which can be troublesome and expensive are avoided by particle analyzing instrumentation solely operated on this Coulter electronic sensing principle. This Coulter sensing principle was developed and expanded into more sophisticated instrumentation such as the COULTER COUNTER (Registered Trademark) Model S types of instruments which enabled CBC parameters, absolute cell counts, platelet count and morphology, red blood cell (RBC) morphology, interpretation of normal and abnormal blood specimens by specific computer programs. The Coulter electronic particle sensing principle employs an aperture sensing circuit using a direct current (DC) aperture supply. Such particle sensors are simple in structure, extremely rugged and reliable as attested to by the substantially universal acceptance of the COULTER COUNTER (Registered Trademark) automated analyzer in clinical laboratories in the United States and throughout the rest of the World. An improvement in this basic aperture sensing circuit was disclosed in U.S. Patent No. 3,502,974 issued in 1970 to Wallace Coulter and Walter Hogg. In addition to the standard direct current aperture supply, a high frequency aperture current was applied which enabled the sensing of an additional parameter for classification purposes. The high frequency aperture current produced a signal which is the function of the blood cell's internal conductivity as well as its volume. The signal produced simultaneously by the direct current aperture circuit is a conventional DC amplitude signal which provides an indication primarily of cell volume. The radio frequency amplitude is divided by the direct current pulse amplitude employing a high speed divider circuit to obtain a quotient which is a function of cell volume and internal resistance, conveniently referred to as "opacity". This principle is further described in U.S. Patent No. 3,502,973 also issued to Wallace Coulter and Walter Hogg, in 1970. This parameter has applicability in cell classification systems. Either a single or a pair of separate apertures could be utilized for this purpose. Classification of different populations is accomplished by collating the data of the signal pairs as they are produced; one, a measure of particle volume and the other a measure of cell internal resistivity or opacity. A convenient form of presenting this data is by two-dimensional plots referred to as scatterplots or scattergrams. Such plots are well described in Flow Cytometry and Sortinσ. page 371; edited by Melamed Melaney, and Medelsohn, 1979, John Wiley & Sons, NY, NY. FIG. 5A is one example of a data plot of a sample of normal blood. Each dot represents an individual cell. The height above the baseline represents the relative volume of the cell. The distance of the dot to the right of the vertical baseline represents the relative opacity. A plot of normal white blood cells (WBC) (with the red blood cells removed) shows three clusters of dots representing three distinct populations which are a consequence of their intrinsic differences in size and internal composition. If desired, with suitable circuitry, these populations can be enumerated to obtain the numbers of each. The cells are classified on the basis of these inherent differences. Initial applications of the Coulter electronic particle sensing principle was to perform red blood cell counts and then, more sophisticated determinations of other red blood cell parameters. By removing red blood cells from whole peripheral blood, analysis of the white blood cell populations could be undertaken so long as the red blood cell removal did not significantly impair properties of the remaining white blood cell populations sought to be measured. Red blood cell lysing reagents were developed for this purpose which, though useful and widely applied, were not entirely satisfactory in all respects for subsequent white blood cell determinations. Previous methods of flow analysis of leukocytes using DC volume alone or light scatter at various angles have shown three clusters of leukocytes corresponding to lymphocytes, monocytes and granulocytes which included the neutrophil, basophil and eosinophil populations. A rough but useful estimation of eosinophil concentration can be made on some samples. The fifth major population is relatively too small for this approach. The eosinophils also have been observed as a distinct cluster using special fluorescence techniques. These fluorescent techniques were utilized in flow cytometry instruments such as the EPICS (Registered Trademark) flow cytometer available from the Coulter Corporation. Such instruments employed the principle of cells moving in a columnar stream bounded by a sheath flow such that cells lined up in single file and passed individually through a laser beam. Light scatter and/or fluorescence signals from the cells were then utilized in classifying cell populations. Staining cells with absorptive or fluorescent dyes made additional cell population classifications possible. The development of instrumentation and fluorochromes for automated multiparameter analysis is further described by R. C. Leif, et al. in Clinical Chemistry, Vol. 23, pp. 1492-98 (1977) . These developments expanded the number of simultaneous population classifications of leukocytes to four, namely lymphocytes, monocytes. eosinophils and "granulocytes" (neutrophils and basophils) . A more recent analytical hematology instrument has utilized light scattering techniques together with peroxidase enzyme staining (absorptive dye) of cells to produce a five part leukocyte differential. Moreover, dyes in combination with specific reacting biological molecules, such as monoclonal antibodies, have increased the number of leukocyte classifications possible to include functional subdivisions. An improved single automated instrument and methods of using the same is disclosed in parent application U.S. Serial No. 587,646, filed September 20, 1990, entitled AUTOMATED ANALYZER AND METHOD FOR SCREENING CELLS OR FORMED BODIES FOR ENUMERATION OF POPULATIONS EXPRESSING SELECTED CHARACTERISTICS. which is a continuation of U.S. Serial No. 025,345, filed March 13, 1987 of the same title. This application combines the application of electronic sensing aperture principles, the specificity of selected biological molecules for identifying and/or enumerating defined populations of cells or formed bodies and microscopic particle technology. The automated analyzer can be used together with a special lysing reagent and/or antibodies coupled to microscopic microspheres or supports of varying composition. A second application, U.S. Serial No. 285,856, filed December 16, 1988, entitled METHOD AND APPARATUS FOR SCREENING CELLS OR FORMED BODIES WITH POPULATIONS EXPRESSING SELECTED CHARACTERISTICS. discloses the screening of direct subsets from whole blood samples or portions thereof. A third application, U.S. Serial No. 339,156, filed April 14, 1989, entitled METHOD AND APPARATUS FOR SCREENING CELLS OR FORMED BODIES WITH POPULATIONS EXPRESSING SELECTED CHARACTERISTICS UTILIZING AT LEAST ONE SENSING PARAMETER, discloses multipart or five part white blood cell differentials, lymphocyte subsets and overlapping determinations performed from a whole blood sample or from a sample with the red blood cells and/or populations of the white blood cells removed by elimination of populations and/or subsets thereof with one or more light or electronic parameters. A fourth application, U.S. Serial No. 07/525,231, filed May 17, 1990, entitled METHOD AND APPARATUS FOR SCREENING OBSCURED OR PARTIALLY OBSCURED CELLS, discloses an analysis of obscured cells by utilizing microspheres having specific monoclonal antibodies bound thereto to move the sensed characteristics of the obscured cells from one cell population area on a scattergram to another area. Each of the four above referenced applications is incorporated herein by reference. An improved analytical hematology instrument and methods of utilizing the same are disclosed in U.S. Serial No. 025,442 filed March 13, 1987 and continuing U.S. Serial No. 129,954 filed December 4, 1987, both entitled MULTI-PART DIFFERENTIAL ANALYZING APPARATUS UTILIZING LIGHT SCATTER TECHNIQUES and are incorporated herein by reference. This so called "VCS" hematology instrument utilizes light scattering and electronic sensing techniques to obtain a multi¬ part differentiation of the leukocyte (L) WBC population. This hematology instrument, however, does not perform differentiation of L subsets, since such subsets are obscured in the L population. Selectively attaching microscopic particles to each cell of a cell population makes possible the modification of the parameter(s) responsible for the original location of at least one of the populations. The addition of a plurality of microscopic particles to each cell of selected target populations where this addition affects the measured volume and/or opacity results in shifting the location of the dots in the scattergram representing a population. Antibodies of known specificity are employed in coating microscopic particles. This coating gives the particle the capacity to selectively attach to certain cells which express the antigen the antibody is specific for. These coated or tagged cells are a combination of particles and a cell which behave like a new entity. Their parameters of opacity, volume, or both opacity and volume may be considered to represent the sum of the effects of both the cell and the particles on the signals obtained. If the characteristics of the components are different, the new entity will move to a new position on a scattergram in accordance with the net effect. The new location, in contrast with the former position of the cell alone, should allow a classification of such new entity or group of new entities. If the particles attached to the cells are magnetic, then, of course, according to current practice, the new entities can be captured by the use of a magnet. If mixed rapidly, unexpected results including complete capture of a population without adversely affecting the properties of the cells under study occur. Only three distinct populations of cells can be readily ident. _.ed and enumerated from a blood sample by utilizing their inherent and unique properties of DC volume and opacity parameters heretofore stated. Additional steps such as improved lysing systems, must be taken to enable the detection and enumeration of more populations. Of course, these additional populations represent subpopulations of the three basic ones referred to as lymphocytes, monocytes and granulocytes. The steps performed in accordance with the above referenced applications demonstrate how subpopulations of these basic three populations are obtained. Employing such simple aperture sensing techniques in combination with two or more biological particles, one can produce a unique and new position of the dot cluster representing a given population. This selective movement of populations on the dot plot or scattergram is reproducible and can be used to classify a population separate from the basic three populations. The original and inherent combination of DC volume and opacity sensing techniques can be modified through the attachment of microscopic particles to selected individual cells. The selectivity is given the particles by the nature or specificity of the biological molecules, antibodies among others, employed as the coating on the particle surfaces. A population of cells alone, having no particles on their surface, may occupy a dot plot position no different from other populations or subpopulations, and, henceforth, not be distinguishable from one another. The addition of particles having a selective attraction to a specific population of cells which one seeks to identify, enumerate, and study is possible using this approach. The selective addition of a sufficient mass of selective particles to a distinct population of interest results in the shifting of that population's dot plot location as a result of the new and unique combination of mass, volume and opacity of each cell. Although the term "reactant" has been utilized in some of the above applications to define lysing agents and monoclonal antibodies, reactants can include various agents which detect and react with one or more specific molecules which are on the surface of a cell. Some examples are given below: Reactant j. Specific Molecule Antibody Antigen Drug Drug Receptor Hormone Hormone Receptor Growth Factor Growth Factor Receptor The reactants couple or bind to the specific molecule(s) on the cells. These reactants do form part of a chemical reaction; however, the reactants are not necessarily chemically altered. One of the most valuable features of this invention is that it employs the simple rugged Coulter sensing operation. It is stable and does not require the complexity and expense of complex optical systems. The circuitry required for the addition of the RF generator and detector is economical, compact and reliable. A single aperture is all that is required, but the additior of a second or even a third aperture can enable a greater sample throughput rate economically. Disclosure of Invention The invention provides a method and apparatus for performing screening of cells in an aged whole blood sample or portion thereof to obtain a multi-part white blood cell population differential. The total white blood cell populations and lymphocyte population are first electronically counted and then the neutrophil population including the aged neutrophil population is removed from the same or a second sample portion. The remaining white blood cell population of monocytes, basophils, lymphocytes and eosinophils including the aged eosinophils then are electronically counted and the two counts are compared to obtain a five-part white blood cell differential. Brief Description of Drawings The preferred embodiments of this invention will now be described by way of example, with reference to the drawings accompanying this specification in which: FIGS. 1-13 describe the embodiments disclosed in the parent application Serial No. 587,646; FIG. 1 is a schematic block diagram of one cell population analyzer embodiment of the parent application; FIG. 2 is a schematic block diagram of a second analyzer embodiment of the parent application; FIG. 3 is one specific analyzer embodiment of the parent application corresponding to FIGS, l and 2; FIG. 4 is a schematic block diagram of another analyzer embodiment of the parent application; FIGS. 5A and 5B are a scattergram of one set of results utilizing a prototype analyzer system similar to that illustrated with respect to FIGS. 2 and 3; FIG. 6 is a schematic block diagram of a further analyzer embodiment of the parent application; FIG. 7 is a schematic block diagram of a still further analyzer embodiment of the parent application; FIGS. 8A and 8B, 9A and 9B, 10A and 10B and 11A and 11B are a scattergram of one set of results utilizing a prototype analyzer system similar to that illustrated with respect to FIGS. 6 and 7; FIG. 12 is a schematic block diagram of a yet still further analyzer embodiment of the parent application; FIG. 13 is a scattergram of one set of results utilizing a prototype analyzer system similar to that illustrated with respect to FIG. 12; FIGURES 14 and 15 describe the embodiments disclosed in the present invention; FIGS. 14A-C are scattergrams of a 24-hour aged sample; and FIGS. 15A-C are scattergrams of a 36-hour aged sample. Mode For Carrying Out The Invention FIGS. 1-13 describe the embodiments of the parent application. Serial No. 587,646. Referring to FIG. 1, a first embodiment of a cell population analyzing method and apparatus of the parent application. Serial No. 587,646, is designated generally by the reference numeral 10. The analyzer 10 includes a biological sample 12 which contains at least a first set of viable biological cells (not illustrated) , such as in or from a whole blood sample. The cells of the biological sample 12 are to be involved in a biological reaction in a quantitative and/or qualitative determination or analysis. The sample 12 can include a buffer into which the cells are added. The sample 12 is combined via a line 14 with at least one reactant 16 via a line 18. The red blood cells (RBC) then are removed from the mixture by a functionally designated RBC removing station 20. The RBC's can be removed from the mixture by the station 20 in a number of ways. The RBC's can be lysed by a lyse in the reactant 16. One such preferential lyse and a quench which can be utilized therewith is disclosed in Serial No. 130,911, filed December 10, 1987, entitled METHOD AND REAGENT SYSTEM FOR ISOLATION. IDENTIFICATION AND/OR ANALYSIS OF LEUKOCYTES FROM WHOLE BLOOD SAMPLES, which is a CIP of Serial No. 025,303, filed March 13, 1987, of the same title, which are incorporated herein by reference. The reactant 16 can be or include a plurality of magnetic microspheres with an antibody specific to the RBC's bound to the microspheres (not illustrated) . In this example, the particular red blood cell specific antibody utilized is disclosed in U.S. Patent No. 4,752,563, entitled MONOCLONAL ANTIBθr τr FOR RECOVERY OF LEUKOCYTES IN HUMAN PERIPHERAL BLOOD AND METHOD OF RECOVERY EMPLOYING SAID MONOCLONAL ANTIBODY, which is incorporated herein by reference. The reactant 16 also can include a buffer in addition to or in place of the sample buffer. The reactant 16 further can be a combination of the preferential RBC lyse and the RBC specific microspheres. Once the RBC's substantially are removed from the mixture, a portion of the mixture is fed into a white blood cell (WBC) analyzer 22 via a line 24. The WBC analyzer 22 at least counts the number of WBC's in the mixture. The WBC analyzer 22 also can measure one or more volume or opacity parameters of the WBC's. The results from the analyzer 22 are fed to a comparator 26 via a line 28. A second portion of the RBC deleted mixture is fed to a WBC subset subtracting station 30 via a line 32. The WBC's can be subtracted from the mixture in a number of ways. Microspheres with a monoclonal antibody specific to one of the WBC subsets bound thereto can be added to the mixture. Non-magnetic or magnetic microspheres can be bound to the WBC's to change or shift the resultant opacity or volume parameters of the cells. Magnetic microspheres also can be bound to the WBC's which then can be removed from the mixture by a magnetic field. The mixture with the WBC subset population removed or with one or more parameters changed then is fed to a WBC subset analyzer 34 via a line 36. The analyzer 34 can be identical to the analyzer 22. The results of the analyzer 34 are then fed to the comparator 26 via a line 38. The comparator 26 then can compare the WBC results from the analyzer 22 with the modified results from the analyzer 34 to determine at least one characteristic of the selected white blood cell population, such as the number of cells in a particular range. Referring to FIG. 2, a second embodiment of a cell population analyzing method and apparatus embodying the parent application is designated generally by the reference numeral 40. The analyzer 40 includes a biological sample 42 which again contains at least a first set of viable biological cells (not illustrated) , such as in or from a whole blood sample. The cells of the biological sample 42 are to be involved in a biological reaction in a quantitative and/or qualitative determination or analysis. The sample 42 again can include a buffer into which the cells are added. The sample 42 is combined via a line 44 with at least one reactant 46 via a line 48. In the analyzer 40, the RBC's are removed from the mixture and simultaneously at least one characteristic of at least one WBC subset is changed or shifted by a functionally designated RBC removing and WBC shifting station 50. As stated above, the RBC's can be removed from the mixture by the station in a number of ways, previously enumerated with respect to the station 20. Simultaneously, in the same mixture portion, the WBC's are bound to, generally non-magnetic, microspheres to change or shift the resultant opacity and/or volume parameters of the cells. The mixture with the RBC's removed and the WBC subset population shifted then is fed to an analyzer 52 via a line 54. The analyzer 52 can be substantially identical to the analyzer 22. The analyzer 40 th..^ provides a fast, direct analysis of at least one- characteristic of a selected WBC population or WBC subset. One specific embodiment of an analyzer instrument embodying the parent application and which can accomplish the analyzing methods of the first and second analyzers 10 and 40, is designated generally by the reference numeral 56 in FIG. 3. In the instrument 56, only one specific enumeration is illustrated, which can be varied in almost endless detail in accordance with the principles of the parent application. Further, the instrument 56 is shown in generally functional detail and the specific embodiments can be structurally implemented in many known ways. The instrument 56 includes an aspirator pumping mechanism 58 which is utilized to draw the biological sample of interest, for example the sample 12 or 42 into the instrument 56. The aspirator 58 is coupled via a line 60 to a sampling valve 62, which can be coupled to a sample probe 63. A diluent pump 64 can include a diluent or buffer solution and also is coupled to the valve 62 via a line 66. The valve 62 and the pump 58 can aspirate the biological sample 12 or 42 along with the diluent via the pump 64 when appropriate. The reactant mixture or the biological sample itself, then is fed via a discharge line 68 into a mixing apparatus 70. The mixer 70 includes a mixing chamber 72 into which the sample or reactant is fed. The RBC's then will be lysed by a lyse from a lyse pump 73 fed into the chamber 72 via a line 74. At this point the operation of the analyzer 10 and 40 differ and hence will be described separately. In the case of the analyzer 10, if the RBC's have been lysed by the lyse from the pump 73, then when the reaction is completed a quench or fix is supplied from a station 75 via a line 76. The reaction can be assisted by mixing the lyse and the sample in the chamber 72 as illustrated functionally at 78. Specific details of an appropriate mixing apparatus 70, which can be utilized herein are disclosed in Serial No. 517,309, filed May 1, 1990, which is a continuation of Serial No. 025,337, filed March 13, 1987, entitled METHOD AND APPARATUS FOR RAPID MIXING OF SMALL VOLUMES FOR ENHANCING BIOLOGICAL REACTIONS. which are incorporated herein by reference. By utilizing the mixer 70 the reactions are greatly enhanced in speed without significantly damaging the properties of interest of the cells, such as, can occur by raising the reaction temperature. Further, the reactions generally are completed in significantly less than a minute, generally on the order of fifteen seconds or less. This allows a rapid analysis of the automatic high volume analyzer instrument 56. The quenched reactant with the RBC's removed by the lyse (as from the station 20) then is fed via a line 80 to a holding chamber 82, which in this case will hold a second portion of the mixture. A first portion of the mixture will be fed from the chamber 82 via a line 84 to a WBC analyzer 86 (i.e., analyzer 22) . The analyzer 86 can be of many physical types in accordance with the counting and sizing techniques described by Wallace H. Coulter in U.S. Patent ' No. 2,656,508 and embodied in the numerous commercial blood cell counters of the assignee. Coulter Electronics, Inc. The analyzer 86, in general, includes a flow sensor or sensing chamber 88. The chamber 88 includes a transducer 90 which has an aperture 92 therethrough. The chamber 88 includes a first portion 94 which has a first electrode 96 in contact with the fluid therein. The chamber portion 94 and the electrode 96 communicate through the aperture 92 with a second chamber portion 98 having a second electrode 100 therein. The electrodes 96 and 100 are coupled via reactive leads 102 and 104 to an RF/DC source and sensing circuit 106. The circuit 106 couples both a DC, or low frequency current or signal, and a high frequency signal between the electrodes 96 and 100. The low frequency signal is utilized to sense the amplitude of a signal pulse caused by a cell passing through the aperture 92. The high frequency signal is utilized to obtain the electrical opacity of the same cell passing through the aperture 92. The measuring of the electrical opacity of cells was described by Wallace H. Coulter and Walter R. Hogg in U.S. Patent No. 3,502,974 and several patents and publications of the assignee. Coulter Electronics, Inc. , since that patent. One specific circuit which can be utilized herein is disclosed in U.S. Patent No. 4,791,355 entitled PARTICLE ANALYZER FOR MEASURING THE RESISTANCE AND REACTANCE OF A PARTICLE, which is incorporated herein by reference. The signals generated by the circuit 106 from the sensed cells are coupled via a DC signal lead 108 and an RF signal lead 110 to a comparator 112 (like the comparator 26) . The comparator 112 can hold the signal generated from the first portion, i.e., those without the WBC subset subtracted, for a comparison with the results from the second portion to be described. The analyzer 86 can include a sheath flow to focus the cells in the sensor 88, in the well known manner. The sheath flow can be provided by a fluidic system 114, coupled to the sensor 88 by a pair of lines 116 and 118 in a known manner. The sample reaction mixture can be fed into the sensor 88 via an introduction tube 120 and can be fed from the sensor 88 via an exit tube 122 into a waste container 124. While the first portion of the mixture was being analyzed in the analyzer 86, the second portion is held in the chamber 82, while the mixer 72 is cleaned or flushed via a rinse line 126 and exhausted through a waste line 128. Once the chamber 72 is cleaned, the second portion is fed back into the chamber 72 via a line 130. Like the station 30, the WBC subset now is subtracted by adding the WBC microspheres from a station 132 via a line 134, a valve 136 and a chamber line 138. The WBC microspheres are mixed with the second portion by the mixing mechanism 78. The reaction mixture with the bound WBC microspheres is fed via the line 80, the chamber 82 and the line 84 into the analyzer 86 (i.e., the analyzer 34), wherein the second portion is analyzed like the first portion and the results then are compared in the comparator 112 (i.e., the comparator 26). At least one of the WBC subset cell parameters is changed in the second portion, such as the cell opacity by the WBC subset bound microspheres to provide the changed results which then can be analyzed. By utilizing magnetic WBC miπrospheres, then the WBC subset bound thereto can be removed by a magnetic field during and/or after the mixing process by a magnetic field or magnet 140. The field can be provided by electromagnetic means or by the magnet 140 being physically moved with respect to the chamber 72 to capture the magnetically bound WBC subset. The second portion without the bound WBC subset then is fed via the line 80, the chamber 82 and line 84 to the analyzer 86 in the manner previously described to obtain the analysis (like the analyzer 34) . The instrument 56 then is prepared to take the next sample for the next analysis. The probe 63 can be cleaned by a probe rinse mechanism 142 and the lines and chamber 72 and 82 can be flushed in a conventional manner. Each analysis of the succeeding sample mixture is obtained in a rapid and automatic fashion. The period between the analysis of succeeding sample mixtures can be on the order of minutes or less. In operating the analyzer instrument 56, like the analyzer 40, the reaction mixture with the RBC lyse/reactant 46 and the sample 42 is mixed in the chamber 72 along with non-magnetic WBC microspheres from the station 132, which bind to one of the WBC subsets. The quench 74 is added to the reactive mixture which then is fed via the line 80, the chamber 82 and the line 84 to the WBC analyzer 86 for analysis (i.e., like the analyzer 52). Alternatively to the utilization of the lyse, in either of the analyzers 10 and 40, the sample 12 or 42 can be fed to the mixer 70 via the valve 62 without any lyse. In this case, the RBC's can be removed magnetically by utilizing the microspheres with the RBC specific antibody bound thereto from an RBC microsphere station 144 and fed to the valve 136 via a line 146 and hence to the chamber 70 via the line 138. Where no lyse is utilized, the bound RBC's are magnetically removed by the magnet 140 after mixing in a manner substantially identical to the magnetically bound WBC's described above. Further, in a second case to promote the speed of the reaction, a reaction mixture of the sample with both the RBC lyse and with the RBC magnetic beads can be utilized. The reaction mixture is mixed, the lyse is quenched and the bound RBC's are magnetically removed and then the WBC's are analyzed as previously described. Referring now to FIG. 4, another embodiment of a cell population analyzing method and apparatus embodying the parent application is designated generally by the reference numeral 148. The analyzer 148 includes a biological sample 150 which again contains at least a first set of viable biological cells, such as in or from a whole blood sample. The sample 150 again can include a buffer into which the cells are added. The sample 150 is combined via a line 152 with at least one reactant 154 via a line 156. The RBC's then are removed as above described by a functionally designated RBC removing station 158. The reaction mixture with the RBC's removed is fed via a line 160 into a WBC analyzer 162. The results from the analyzer 162 are fed to a comparator 164 via a line 166, providing a three-part WBC differential with results for monocytes (M) , lymphocytes (L) and granulocytes (G) . The mixture then is fed to a neutrophil (N) functionally designated removal station 168 via a line 170. The N's can be removed from the mixture by shifting or changing one parameter, such as opacity, or by magnetic removal, both as described above. In this example, the particular N specific antibody utilized is disclosed in U.S. Patent No. 4,931,395, MONOCLONAL ANTIBODY SPECIFIC TO NEUTROPHILS. The mixture with the N's removed or shifted then is fed to another WBC analyzer 172 via a line 174. The results of the analyzer 172 are fed to the comparator 164 via a line 176. The results of the analyzer 172 are utilized to obtain a four-part WBC differential with results again for M's and L's, but now in addition since the N's are shifted or removed results for eosinophils (E) and basophils (B) are obtained. The two analytical results from the analyzers 162 and 172 then can be compared by the comparator 164 to form a five-part WBC differential. Specifically, subtracting the number of B's and E's from the number of G's results in the number of the removed N's. Referring now to FIGS. 5A and 5B, two sets of scattergram results are illustrated obtained from a whole blood sample utilizing a prototype analyzing method similar to the analyzer 148. The biological sample 150 was a 20 microliter sample of whole blood which was combined with 40 microliters of the magnetic microspheres with the RBC specific antibody bound thereto combined with 140 microliters of buffer solution to form the reactant 154. The reaction mixture was mixed for 15 seconds and placed in a magnetic field for 10 seconds in the station 158. The mixture with the RBC's removed was analyzed by the analyzer 162 as illustrated in the scattergram of FIG. 5A resulting in counts of L's of 45.6 (1), M's of 5.6 (2) and G's of 48.7 (3). The mixture then is combined in the station 168 with 10 microliters of magnetic microspheres with the N specific antibody bound thereto. The mixture is mixed 30 seconds and then placed in a magnetic field for 10 seconds. The mixture with the N's then removed was fed to the analyzer 176 which resulted in the scattergram of FIG. 5B resulting in counts of L's - of 81.0 (1), M's of 0.6 (2), E's of 11.0 (3) and B's of 1.8 (4). The comparator 164 then provides the five-part WBC differential of counts of 45.6 L's, 5.6 M's, 41.6 N's, 6.0 E's and 1.2 B's. This corresponds to a standard microscopic five-part WBC differential utilizing Wright stain on the sample on a slide resulting in counts of 44.0 L's, 3.4 M's, 45.0 N's, 6.1 E's and 0.4 B's. FIG. 6 illustrates a further embodiment of a cell population analyzing method and apparatus embodying the parent application, designated generally by the reference numeral 178. The analyzer 178 includes a biological sample 180 which again contains at least a first set of viable biological cells and also can include a buffer. The sample 180 is combined via a line 182 with a reactant 184 via a line 186. Functionally illustrated, a first portion of the mixture is fed via a line 188 to a functionally designated RBC and N removing station 190. The RBC's and N's are removed or shifted as described before and the first portion is fed vi; a line 192 to a WBC analyzer 194. This provides a result from the analyzer 194 which is fed via a line 196 to a comparator 198. The results includes the above-referenced four-part differential including M's, L's, E's and B's. At th-i same time, a second portion of the mixture of the sample 180 and the reactant 184 is fed via a line 200 to a functionally designated RBC removal station 202. The mixture with the RBC's removed is fed via a line 204 to another WBC analyzer 206. The results of the analyzer 206 are fed to the comparator 198 via a line 208. The results of the analyzer 206 directly include the above-referenced three-part WBC differential including M's, L's and G's. the results of the analyzers 194 and 206 then are compared by the comparator 198 to provide the five-part WBC differential. A specific analyzing instrument embodiment incorporating the method and apparatus of the analyzer 178 is designated generally by the reference numeral 210 in FIG. 7. Again, only one specific hardware enumeration has been illustrated, but like the analyzing instrument 56, the analyzing instrument 210 can be implemented in numerous configurations. The instrument 210 includes an aspirator purging mechanism 212 which is coupled to a sampling valve 214 via a line 216. The valve 214 can include a sample probe 218 to aspirate the biological sample of interest, such as the sample 180. A diluent delivery pump 220 is coupled to the valve 214 via a line 222 to provide a diluent for the sample, such as a whole blood sample, when desired. A first portion of the mixture then is coupled via a line 224 and a line 226 to a first mixing apparatus 228. At the same time, a second portion of the mixture is fed via the line 224 and a line 230 to a second mixing apparatus 232. The mixer 228 (comparable to the station 190) is substantially identical to the mixer 232 (comparable to the station 202) and will be described first. The mixer 228 includes a mixing chamber 234 into which the first mixture portion is fed. The mixer 228 includes all of the various options above described and can include a lyse input line 236 for the RBC lyse if desired. If the lyse is utilized, after mixing as illustrated functionally at 238, then the quench is added via a quench line 240. At the same time, the N's are being removed by the addition of the appropriate magnetic or non-magnetic microspheres with the N specific antibody bound thereto from a source of microspheres 242 fed to the chamber 234 via a line 244. If magnetic microspheres are utilized for the N's or the RBC's, then a magnet 246 or magnetic field is utilized to remove the magnetically bound cells. The mixed and quenched (where necessary) mixture then is fed via a line 248 through a valve 250 and a line 252 to a WBC analyzer 254 (i.e., analyzer 194). The analyzer 254 is the same as the analyzer 86 and will not be described again in such detail. Again, the analyzer 254 includes a sensing chamber 256 with an aperture 258 therein through which the mixture and cells pass. A sheath flow fluidic system 260 can be coupled to the chamber 256. The signals generated by the cells are detected by an RF/DC source and sensing circuit 262 whose outputs are fed to a comparator 264, as previously described. Concurrently, the second mixture portion is fed into a mixing chamber 266. In the second portion, only the RBC's are removed (i.e., like the station 202) and the RBC's can be removed by the RBC lyse fed into the chamber 266 via a line 268. The lyse is mixed with the sample and then a quench is added via a quench line 270. Alternatively the RBC's can be removed by magnetic microspheres having the RBC specific antibody bound thereto from a microsphere source 272 fed into the chamber 266 via a line 274. The microspheres are mixed, functionally at 276, and then the magnetically bound RBC microspheres are removed by a magnet 278. The RBC removed mixture then is fed via a line 280 to the valve 250 and via the line 252 to the analyzer 254 to obtain the above-mentioned results. The mixers 228 and 232 include appropriate respective rinse lines 282 and 284 and waste lines 286 and 288 and a probe rinse 290 to cleanse the instrument 210 ■ prior to aspirating the next sample or sample for analyzing. FIGS. 8A and 8B illustrate scattergram results obtained from a whole blood sample utilizing an analyzing method similar to the analyzer 178. In this example, 20 microliters of whole blood form the sample 180, while 40 microliters of magnetic microspheres with the RBC specific antibody bound thereto combined with 140 microliters of buffer solution form the reactant 184. A portion of the mixture is mixed for 20 seconds in the station 202 and then placed in a magnetic field for 10 seconds. The RBC removed mixture then is analyzed in the analyzer 206 resulting in the scattergram of FIG. 8A which provides a count of L's 29.4 (1), M's 8.1 (2) and G's 62.4 (3) . At the same time, another portion of the same mixture is combined with 10 microliters of magnetic microspheres with the N specific antibody bound thereto to remove the RBC's and N's in the station 190. The mixture is mixed for 30 seconds, then placed in a magnetic field for 10 seconds. The mixture with the N's and RBC's removed then is analyzed by the analyzer 194 resulting in the scattergram of FIG. 8B which provides a count of L'S 73.5 (1), M'S 21.7 (2), E's 3.4 (3) and B's 1.4 (4) . The two counts are compared in the comparator 198, resulting in a five-part WBC differential count of L's 29.4, M's 8.0, N's 60.8, E's 1.2 and B's 0.6. A microscope comparison again was made resulting in counts of L's 29.4, M's 5.0, N's 65.0, E's 1.0 and B's of less than 1.0. FIGS. 9A and 9B show scattergram results of a five-part WBC differential example similar to that of FIGS. 8A and 8B. A 20 microli er sample of whole blood was analyzed in the same steps described with * respect to FIGS. 8A and 8B resulting in the scattergram of FIG. 9A providing a count of L's 35.4 (1), M's 14.6 (2) and G's 50.0 (3). The scattergram of FIG. 9B provides a count of L'S 66.4 (1), M's 25.0 (2), E's 6.6 (3) and B's 2.0 (4). The resulting five-part WBC differential results in counts of 35.4 L's, 14.6 M's, 45.5 N's, 3.5 E's and 1.1 B's was compared to a microscope count of 36 L's, 11 M's, 49 N's, 3 E's and 1 B. FIGS. 10A and 10B show scattergram results of a five-part WBC differential again similar to that of FIGS. 8A, 8B and 9A, 9B, however, in this example, lyse was utilized. In this example, 20 microliters of whole blood was combined with 80 microliters of buffer and 240 microliters of the RBC preferential lyse above referenced. The mixture is mixed for 6 seconds and then a quench is added. The time period is significant, because the lyse left unquenched for a period of time greater than about 10 seconds will start to affect the significant properties of the WBC's. The mixture with the RBC's removed is analyzed to provide the scattergram of FIG. 10A resulting in counts of L's 25.7 (1), M's 9.6 (2) and G's 65.0 (3) . A second portion of the mixture including a second 20 microliter sample of the whole blood is combined with 120 microliters of buffer and 10 microliters of magnetic microspheres with the N specific antibody bound thereto and mixed for 30 seconds and then placed in a magnetic field for 10 seconds. The RBC preferential lyse then is added to the N removed mixture which then is mixed for 6 seconds before it is quenched. The resulting scattergram FIG. 10B results in percentage counts of L's 74.6 (1), M's 21.6 (2), E's 2.9 (3) and B's 0.8 (4). The resulting five-part WBC differential results in percentage counts of L's 25.6, M's 9.6, N's 63.5, E's 1.06 and B's 0.3. Again a microscope comparison resulted in counts of L's 29.4, M's 5.0, N's 65.0, E's 1.0 and B's of less than 1. Another example of scattergram results of a five-part WBC differential similar to that of FIGS. 10A and 10B is illustrated in FIGS. 11A and 11B. A sample of whole blood had two samples simultaneously analyzed in the same steps described with a respect to FIGS. 10A and 10B. The scattergram of FIG. 11A provides a count of L's 31.9 (1), M's 17.6 (2) and G's 50.4 (3). The scattergram of FIG. 11B provides a count of L's 67.1 (1), M's 24.1 (2), E's 7.6 (3) and B's 1.2 (4). The resulting five-part WBC differential results in counts of 31.9 L's, 11.4 M's, 46.0 N's, 3.6 E's and 0.7 B's as compared to a microscope count of 36 L's, 11 M's, 49 N's, 3 E's and 1 B's. A yet still further embodiment of a cell population analyzing method and apparatus embodying the parent application is designated generally by the reference numeral 292 in FIG. 12. The analyzer 292 includes a biological sample 294, again including at least a first set of viable biological cells and including a buffer if desired. The sample 294 is combined via a line 296 with at least one reactant 298 via a line 300. In the analyzer 292, the RBC's are removed and the N's are shifted sequentially or simultaneously in a functionally designated station 302. The RBC remove function is designated 304 and the N move or shift portion is designated 306 to indicate that the functions can be performed simultaneously or sequentially. The RBC's can be removed magnetically - or with lyse or with a combination of the two as previously described. The N's are removed or shifted by adding microspheres having an N specific antibody bound thereto to the mixture. Once the RBC's are removed and the N's are moved or shifted, then the resulting mixture is fed via a line 308 to an analyzer 310. In this case, the N's are shifted sufficiently from the patterns of the E's and B's that a five-part WBC differential of M's, L's, E's, B's and N's is directly obtained. The functions of the analyzer 292 can be performed on either of the instruments 56 and 210 or minor variations thereof. The scattergram results of one example of a direct five-part WBC differential in accordance with the analyzer 292 is illustrated in FIG. 13. In this example, the biological sample 294 is 20 microliters of a whole blood sample and the reactant 298 is 10 microliters of non-magnetic microspheres with the N specific antibody bound thereto combined with 100 microliters of buffer and mixed in the substation 306 for 30 seconds. The RBC preferential lyse, 10 microliters thereof, then is added to the mixture which is mixed in the substation 304 for 6 seconds after which the quench is added. The RBC removed and N shifted mixture then is analyzed by the analyzer 310 resulting in the scattergram of FIG. 13 which provides a direct count of 29.6 L's, 13.6 M's, 52.2 N's, 3.4 E's and 1.06 B's as compared to a microscope determination of 35 L's, 5 M's, 56 N's, 4 E's and no B's. In this particular example, the whole blood sample was also analyzed on a general cell counting instrument of Coulter Electronics, Inc., which resulted in 29 L's, 11.1 M's and 59.9 G's (N's, E's and B's) . * Referring now to FIGS. 14 and 15, the embodiments of the present invention are illustrated. The embodiments of the WBC population analyzer methods and apparatus of the present invention can be substantially the same as the analyzers 56, 148 and 178 illustrated in FIGS. 3, 4 and 6. The analyzers include a biological sample, which contains at least a first set of viable biological cells (not illustrated) , including the white blood cell populations. Applicant has discovered that the multi-part differential procedure of the parent application operates efficiently and accurately in a typical hospital operation, where the blood sample is on the order of 12 hours old or less. However, when the blood sample is in the range of 18-24 hours old or older, the age of the sample causes differences in the location of the cell data points on the scattergram. For example a normal blood sample scattergram utilizing the electronics sensing parameters of DC and opacity generate patterns like FIGS. 9A, 10A and 11A. The N's are removed from the blood sample resulting in patterns of L's, M's, E's and B's as shown in FIGS. 9B, 10B and 11B. The parent application N removal procedure thus provided the desired five-part WBC differential. However, in an aged blood sample, such as illustrated in FIGS. 14A and 15A, the scattergrams are no longer clearly identifiable as L's, G's and M's. The procedure of the parent application produces no or erroneous results when utilized with the aged samples, FIG. 14A illustrating a blood sample about 26 hours old and FIG. 15A illustrating a blood sample about 36 hours old. These aged samples produce patterns which are not readily identifiable, apparently because of a new population or populations, as can be seen by comparing FIGS. 9A, 10A and 11A with FIGS. 14A and 15A. Referring first to FIG. 14A, Applicant first utilized the electronic sensing parameters of the parent application to obtain the scattergram pattern 350. This pattern 350 was not the same as for a normal (non-aged) blood sample, because as can be seen by comparing, for example FIGS. 9A, 10A or 11A with FIG. 14A, a new cell grouping 354 now appears in the aged sample scattergram. Applicant then removed the N's to obtain a scattergram, 356 illustrated in FIG. 14C, also in accordance with the parent application. It appeared that some of the cells in the cell grouping 354 were aged N's, which were removed with the other N's. This however did not allow for a proper five-part differential since some unidentified cells still remained to form a cell grouping 358. It appeared that the scattergram 356 included separate L, M, E and B groupings, but that still left the grouping 358 unaccounted for. Since this technique did not allow a five-part white blood cell differential to be obtained in accordance with the parent application. Applicant then utilized the light scatter techniques of the VCS type instrument, above referenced to obtain another pattern 352, as illustrated in FIG. 14B. The scattergram 352 also could not be analyzed utilizing the normal VCS techniques, again because of the new undefined population 354. Applicant then attempted to determine what cell or cells were causing the scattergram pattern to change with age. To determine what the cells in the grouping 358 could be. Applicant analyzed the pattern 356. Since it appeared that some aged N's were in the pattern 354, Applicant decided to determine if the pattern 358 was caused by aged E's. Applicant first added the cell count in the pattern 358 to the number of E's and compared this to a normal (non- aged) five-part differential for this sample (previously obtained) . This indeed corrected the erroneous five-part differential results, confirming that the remaining cells in the pattern 358 were aged E's. Applicant then utilized an N and E specific antibody disclosed in U.S. Serial No. 068,618, entitled MONOCLONAL ANTIBODY SPECIFIC TO A COMMON DETERMINANT SITE OF NEUTROPHILS AND EOSINOPHILS. filed June 13, 1987, which is incorporated herein by reference. Since it appeared that the N's already were removed, the antibody again bound to magnetic microspheres was expected to remove at least all of the E's. The microspheres removed both all of the E grouping and the cell grouping 358. This again confirmed that the aged cells in the scattergram 350 were both aged E's and aged N's. The cells in grouping 358 also could be aged N's that no longer display an affinity to the N specific antibody, but still display an affinity to the N and E specific antibody. However, since the final results are compared back to the zero time results and the number of E's is correct, this suggests the remaining cells are aged E's and not aged N's. If the cells were aged E's and aged N's as appeared, then the technique of the parent application could be applied, with the following modifications. The total WBC populations and the L population are first counted, including all of the grouping 354. Unlike the scattergram of the samples illustrated in FIGS. 9A, 10A and 11A, the G's and M's are not separately identifiable in the scattergram 350. Therefor, the total WBC populations are counted as indicated by and included in a block 360. The L's appeared identifiable in both of the sca tergrams 350 and 356 and hence they are separately counted as indicated by a block 362 in FIG. 14A. The N's then are removed which removes both the N's from the E's area in the scattergram 356 of FIG. 14C as well as the aged N's from the grouping 354. The L's, M's, B's and E's then can be counted in the scattergram 356. The L's are correlated with the L's in the block 362. The total WBC populations includes the block or grouping 358. A count for the removed N's is obtained by comparing the two total WBC counts. The L and total WBC counts can be utilized to correlate the individual M, B and E counts. The E's total counts include the E grouping and the aged E 358 grouping. Applicant also has determined that the errors caused by the aging of the sample cannot be precisely predicted as to time of occurrence. However, the errors appear to occur on a one cell to one cell basis and hence the technique of the present invention can be utilized at any time. In the case of a normal pattern such as shown in FIGS. 9A, 10A and 10B, the count for the groupings 354 and 358 will be zero. The technique thus ensures a correct five- part differential, whether or not there are any aged cells. To confirm the results of the technique of the present invention, samples were tested utilizing the VCS instrument at zero hours aging which are compared to some aged 24 hour samples utilizing the present invention. The results compare very well as illustrated by Table I. The 24 hour sample results in Table I all were obtained utilizing electronic RF and DC sensing parameters. To confirm that the present invention also can be utilized with light scatter, such as in the VCS instrument, one set of 24 hour sample data was obtained as illustrated in Table II. TABLE II The techniques of the present invention also were applied to 36 hour aged samples as illustrated in FIGS. 15A-C. Another scattergram 364 produced by electronic sensing is illustrated in FIG. 15A, while a VCS scattergram 366 is illustrated in FIG. 15B. Again, the total white blood cell populations are counted as indicated by a block or grouping 368 and the L's also are counted as indicated by a block or grouping 370. The N's then are removed as before, including the aged N's resulting in a scattergram 372, which again includes the L's, M's, E's, B's and aged E's in a grouping 374. Results from the 36 hour aged samples can be compared with the VCS zero age data in Table III. TABLE III Patient: The magnetic microspheres utilized can be of any suitable type and for example, are polystyrene magnetic microspheres of 0.7 micron diameter, with a weight to volume of 10% solids, sold by Bangs Laboratories of Carmel, Indiana. The non-magnetic microspheres again can be of any suitable type and for example, are surfactant free sulfated polystyrene latex microspheres of 2.17 micron diameter with a weight to volume of 8% solids, sold as IDC microspheres by Interfacial Dynamics of Portland, Oregon. Although these specific microspheres are utilized for example purposes, other types and sizes of microspheres from other conventional sources also can be utilized. In general, for shifting, it is preferable to utilize microspheres of a diameter substantially less than the diameter of the cells, since a plurality of the microspheres should bind to each cell. Typically, microspheres of diameters from 0.65 to 3.0 microns are utilized to ensure that the instrument does not sense and count the free microspheres themselves as an indicated cell. In general, the cell antigen density also affects the size of microspheres utilized, since a low density (binding a few microspheres) would require larger microspheres and a high density (binding numerous microspheres) would allow use of smaller microspheres. If the microspheres are too large the system can count the free microspheres as cells to generate an inaccurate count and hence an inaccurate analysis. Further, very large microspheres could clog or block the sensing apertures, since a plurality of cells could be bound thereto, resulting in a large microsphere and cell mass. Typically, non-magnetic microspheres are less expensive and hence are utilized for shifting purposes, although magnetic microspheres also can be utilized to cause the sensed cell shift. To eliminate cells magnetically, only magnetic microspheres can be utilized. In this application, the microsphere size is less relevant, since the cells are to be eliminated from the sample before sensing. The microspheres should be of sufficient means to quickly and easily be removed in the magnetic field. In this case, 0.65 to 4.5 micron diameter microspheres work well. Further, since the cells only are to be eliminated, 10 or greater micron diameter microspheres could be utilized. A plurality of cells could be bound to each microsphere, but since no counting will take place, this does not interfere with the operation of the instrument.

Claims: CLAIMS 1. A method according to a multi-part white blood cell population differential from at least a portion of a whole blood sample having at least white blood cell populations therein, characterized by: electronically counting at least the total white blood cell populations and the lymphocyte population; removing the neutrophil population including the aged neutrophil population contribution from said white blood cell populations; electronically counting at least said remaining white blood cell populations of monocytes, lymphocytes, eosinophils, aged eosinophils and basophils; and comparing said two counts to obtain a count of said white blood cell population of neutrophils and aged neutrophils and thereby obtaining at least a five-part white blood cell differential. 2. The method according to claim 1 further characterized by said whole blood sample includes a red blood cell population and removing said red blood cell population from said sample without adversely affecting relevant qualities and/or quantities of said white blood cell populations. 3. The method according to claim 2 further characterized by removal of said red blood cell population includes providing microspheres having a red blood cell specific monoclonal antibody bonded thereto and mixing said microspheres with said whole blood sample to bind to said red blood cell population; and removing said microspheres with said bound red blood cells from said whole blood sample. 4. The method according to claim 3 further characterized by providing magnetic microspheres and a magnetic field and removing said microspheres by removing said red blood cells while attracting said magnetic microspheres within said magnetic field. 5. The method according to claim 3 further characterized by rapidly mixing said microspheres with said whole blood sample to bind said red blood cell population to said microspheres in less than sixty seconds. 6. The method according to claim 2 further characterized by removing said red blood cell population includes providing microspheres having a red blood cell specific monoclonal antibody bonded thereto and mixing said microspheres with said whole blood sample to bind to said red blood cell population and providing a red blood cell lyse with said microspheres to eliminate a portion of said red blood cell population to decrease the number of microspheres necessary to remove said red blood cell population; and removing said microspheres with said red blood cells bound thereto from said whole blood sample. 7. The method according to claim 6 further characterized by providing magnetic microspheres and a magnetic field and removing said microspheres by removing said red blood cells while attracting said magnetic microspheres within said magnetic field. 8. The method according to claim 6 further characterized by rapidly mixing said microspheres with said whole blood sample to bind said red blood cell population to said microspheres in less than sixty seconds and to provide said lysing action. 9. The method according to claim 2 further characterized by removing said red blood cell population includes providing a red blood cell lyse to substantially eliminate said red blood cell population. 10. The method according to claim 1 further characterized by removing the neutrophil and aged neutrophil populations includes providing microspheres having a neutrophil specific monoclonal antibody bonded thereto and mixing said microspheres with said sample to bind to said neutrophil populations; and removing said microspheres with said neutrophil populations bound thereto from said sample. 11. The method according to claim 10 further characterized by providing magnetic microspheres and a magnetic field and removing said microspheres by removing said neutrophil populations while attracting said magnetic microspheres within said magnetic field. 12. The method according to claim 10 further characterized by rapidly mixing said microspheres with said sample to bind said neutrophil populations to said microspheres in less than sixty seconds. 13. An apparatus for obtaining a multi-part white blood cell population differential from at least a portion of a whole blood sample having at least white blood cell populations therein, characterized by: means for electronically counting at least the total white blood cell populations and the lymphocyte population; means for removing the neutrophil population including the aged neutrophil population contribution from said white blood cell populations; means for electronically counting at least said remaining white blood cell populations of monocytes, lymphocytes, eosinophils, aged eosinophils and basophils; and means for comparing said two counts to obtain a count of said white blood cell population of neutrophils and aged neutrophils and thereby obtaining at least a five-part white blood cell differential. 14. The apparatus according to claim 13 further characterized by said whole blood sample includes a red blood cell population and means for removing said red blood cell population from said sample without adversely affecting relevant qualities and/or quantities of said white blood cell populations. 15. The apparatus according to claim 14 further characterized by removal of said red blood cell population includes providing microspheres having a red blood cell specific monoclonal antibody bonded thereto and means for mixing said microspheres with said whole blood sample to bind to said red blood cell population; and means for removing said microspheres with said bound red blood cells from said whole blood sample. 16. The apparatus according to claim 15 further characterized by means for providing magnetic microspheres and a magnetic field and removing said microspheres by removing said red blood cells while attracting said magnetic microspheres within said magnetic field. 17. The apparatus according to claim 15 further characterized by means for rapidly Eixing said microspheres with said whole blood sample to bind said red blood cell population to said microspheres in less than sixty seconds. 18. The apparatus according to claim 14 further characterized by said means for removing said red blood cell population include providing microspheres having a red blood cell specific monoclonal antibody bonded thereto and means for mixing said microspheres with said whole, blood sample to bind to said red blood cell population and providing a red blood cell lyse with said microspheres to eliminate a portion of said red blood cell populations to decrease the number of microspheres necessary to remove said red blood cell populations; and means for removing said microspheres with said red blood cells bound thereto from said whole blood sample. 19. The apparatus according to claim 18 further characterized by means for providing magnetic microspheres and a magnetic field and removing said microspheres by removing said red blood cells while attracting said magnetic microspheres within said magnetic field. 20. The apparatus according to claim 18 further characterized by means for rapidly mixing said microspheres with said whole blood sample to bind said red blood cell population to said microspheres in less than sixty seconds and to provide said lysing action. 21. The apparatus according to claim 14 further characterized by said means for removing said red blood cell population includes providing a red blood cell lyse to substantially eliminate said red blood cell population. 22. The apparatus according to claim 13 further characterized by said means for removing the neutrophil and aged neutrophil populations include providing microspheres having a neutrophil specific monoclonal antibody bonded thereto and means for mixing said microspheres with said sample to bind to said neutrophil populations; and means for removing said microspheres with said neutrophil populations bound thereto from said sample. 23. The apparatus according to claim 22 further characterized by means for providing magnetic microspheres and a magnetic field and removing said microspheres by removing said neutrophil populations while attracting said magnetic microspheres within said magnetic field. 24. The apparatus according to claim 22 further characterized by means for rapidly mixing said microspheres with said sample to bind said neutrophil populations to said microspheres in less than sixty seconds. 25. A method of obtaining a multi-part white blood cell population differential from at least a portion of a whole blood sample having at least white blood cell populations therein, characterized by: electronically counting at least the total white blood cell populations and the lymphocyte population; removing the neutrophil population including the aged neutrophil population contribution from said white blood cell populations from a second portion of said sample without adversely affecting the relevant qualities and/or quantities of said remaining white blood cell populations; electronically counting at least said remaining white blood cell populations of monocytes, lymphocytes, eosinophils, aged eosinophils and basophils in said second portion; and comparing said two counts from said first and second portions to obtain a count of said white blood cell populations of neutrophils and aged neutrophils and thereby obtaining at least a five-part white blood cell differential. 26. The method according to claim 25 further characterized by said whole blood sample includes a red blood cell population and removing said red blood cell population from said sample without adversely affecting relevant qualities and/or quantities of said white blood cell populations prior to the counting of said first portion and also removing said red blood cell population from said second portion prior to the counting thereof. 27. The method according to claim 26 further characterized by removing said red blood cell population from at least one of said portions includes providing microspheres having a red blood cell specific monoclonal antibody bonded thereto and mixing said microspheres with said whole blood sample to bind to said red blood cell population; and removing said microspheres with said red blood cells bound thereto from said whole blood sample. 28. The method according to claim 27 further characterized by providing magnetic microspheres and a magnetic field and removing said microspheres by removing said red blood cells while attracting said magnetic microspheres within said magnetic field. 29. The method according to claim 27 further characterized by rapidly mixing said microspheres with said whole blood sample to bind to said red blood cell population to said microspheres in less than sixty seconds. 30. The method according to claim 26 further characterized by removing said red blood cell population from at least one of said portions includes providing microspheres having a red blood cell specific monoclonal antibody bonded thereto and mixing said microspheres with said whole blood sample to bind to said red blood cell population and providing a red blood cell lyse with said microspheres to eliminate a portion of said red blood cell populations to decrease the number of microspheres necessary to remove said red blood cell populations; and removing said microspheres with said red blood cells bound thereto from said whole blood sample. 31. The method according to claim 30 further characterized by providing magnetic microspheres and a magnetic field and removing said microspheres by removing said red blood cells while attracting said magnetic microspheres within said magnetic field. 32. The method according to claim 30 further characterized by rapidly mixing said microspheres with said whole blood sample portion to bind said red blood cell population to said microspheres in less than sixty seconds and to provide said lysing action. 33. The method according to claim 26 further characterized by removing said red blood cell population from at least one of said portions includes providing a red blood cell lyse to substantially eliminate said red blood cell population. 34. The method according to claim 25 further characterized by removing the neutrophil and aged neutrophil populations in said second portion includes providing microspheres having a neutrophil specific monoclonal antibody bonded thereto and mixing said microspheres with said whole blood sample to bind to said neutrophil populations; and removing said microspheres with said neutrophil populations bound thereto from said sample. 35. The method according to claim 34 further characterized by providing magnetic microspheres and a magnetic field and removing said microspheres by removing said neutrophil populations while attracting said magnetic microspheres within said magnetic field. 36. The method according to claim 34 further characterized by rapidly mixing said microspheres with said whole blood sample to bind said neutrophil populatioiiS to said microspheres in less than sixty seconds. 37. An apparatus for obtaining a multi-part white blood cell population differential from at least a portion of a whole blood sample having at least white blood cell populations therein, characterized by: means for electronically counting at least the total white blood cell populations and the lymphocyte population in a first portion of said sample; means for removing the neutrophil population including the aged neutrophil population contribution from said white blood cell populations from a second portion of said sample without adversely affecting the relevant qualities and/or quantities of said remaining white blood cell populations; means for electronically counting at least said remaining white blood cell populations of monocytes, lymphocytes, eosinophils, aged eosinophils and basophils in said second portion; and means for comparing said two counts from said first and second portions to obtain a count of said white blood cell populations of neutrophils and aged neutrophils and thereby obtaining at least a five- part white blood cell differential. 38. The apparatus according to claim 37 further characterized by said whole blood sample includes a red blood cell population and including means for removing said red blood cell population from said sample without adversely affecting relevant qualities and/or quantities of said white blood cell populations prior to the counting of said first portion and also removing said red blood cell population from said second portion prior to the counting thereof. 39. The apparatus according to claim 38 further characterized by said means for removing said red blood cell population from at least one of said portions include providing microspheres having a red blood cell specific monoclonal antibody bonded thereto and means for mixing said microspheres with said whole blood sample to bind to said red blood cell population; and means for removing said microspheres with said red blood cells bound thereto from said whole blood sample. 40. The apparatus according to claim 39 further characterized by means for providing magnetic microspheres and a magnetic field and removing said microspheres by removing said red blood cells while attracting said magnetic microspheres within said magnetic field. 41. The apparatus according to claim 39 further characterized by means for rapidly mixing said microspheres with said whole blood sample to bind to said red blood cell population to said microspheres in less than sixty seconds. 42. The apparatus according to claim 38 further characterized by said means for removing said red blood cell population from at least one of said portions includes providing microspheres having a red blood cell specific monoclonal antibody bonded thereto and means for mixing said microspheres with said whole blood sample to bind to said red blood cell population and providing a red blood cell lyse with said microspheres to eliminate a portion of said red blood cell populations to decrease the number of microspheres necessary to remove said red blood cell populations; and means for removing said microspheres with said red blood cells bound thereto from said whole blood sample. 43. The apparatus according to claim 42 further characterized by means for providing magnetic microspheres and a magnetic field and removing said microspheres by removing said red blood cells while attracting said magnetic microspheres within said magnetic field. 44. The apparatus according to claim 42 further characterized by means for rapidly mixing said microspheres with said whole blood sample portion to bind said red blood cell population to said microspheres in less than sixty seconds and to provide said lysing action. 45. The apparatus according to claim 38 further characterized by said means for removing said red blood cell population from at least one of said portions includes providing a red blood cell lyse to substantially eliminate said red blood cell population. 46. The apparatus according to claim 37 further characterized by said means for removing the neutrophil and aged neutrophil populations in said second portion includes providing microspheres having a neutrophil specific monoclonal antibody bonded thereto and means for mixing said microspheres with said whole blood sample to bind to said neutrophil populations; and means for removing said microspheres with said neutrophil populations bound thereto from said sample. 47. The apparatus according to claim 46 further characterized by means for providing magnetic microspheres and a magnetic field and removing said microspheres by removing said neutrophil populations while attracting said magnetic microspheres within said magnetic field. 48. The apparatus according to claim 46 further characterized by means for rapidly mixing said microspheres with said whole blood sample to bind said neutrophil populations to said microspheres in less than sixty seconds. AMENDED CLAIMS [received by the International Bureau on 12 October 1992 (12.10.92); original claims 10,22,34 and 46 deleted; original claim 1 amended; original claims 11-48 amended and renumbered as claims 10-44; new claims 45 and 46 added; remaining claims unchanged ( 10 pages )] 1. A method of obtaining a not otherwise obtainable multi-part white blood cell population differential from at least a portion of a whole blood sample having at least white blood cell populations therein for identification and/or enumeration and/or study, characterized by: electronically counting at least the total white blood cell populations and the lymphocyte population; removing the neutrophil population including the aged neutrophil population contribution from said white blood cell populations by providing microspheres having a neutrophil specific monoclonal antibody bonded thereto and mixing said microspheres with said sample to bind to said neutrophil populations and removing said microspheres with said neutrophil populations bound thereto from said sample and in so doing allowing for the electronic sensing of eosinophils and basophils; electronically counting at least said remaining white blood cell populations of monocytes, lymphocytes, eosinophils, aged eosinophils and basophils; and comparing said two counts to derive a count of said white blood cell population of neutrophils and aged neutrophils and thereby obtaining at least a five-part white blood cell differential. 2. The method according to claim 1 further characterized by said whole blood sample includes a red blood cell population and removing said red blood cell population from said sample without adversely affecting relevant qualities and/or quantities of said white blood cell populations. 3. The method according to claim 2 further characterized by removal of said red blood cell population includes providing microspheres having a red blood cell specific monoclonal antibody bonded thereto and mixing said microspheres with said whole blood sample to bind to said red blood cell population; and removing said microspheres with said bound red blood cells from said whole blood sample. 4. The method according to claim 3 further characterized by providing magnetic microspheres and a magnetic field and removing said microspheres by removing said red blood cells while attracting said magnetic microspheres within said magnetic field. 5. The method according to claim 3 further characterized by rapidly mixing said microspheres with said whole blood sample to bind said red blood cell population to said microspheres in less than sixty seconds. 6. The method according to claim 2 further characterized by removing said red blood cell population includes providing microspheres having a red blood cell specific monoclonal antibody bonded thereto and mixing said microspheres with said whole blood sample to bind to said red blood cell population and providing a red blood cell lyse with said microspheres to eliminate a portion of said red blood cell population to decrease the number of microspheres necessary to remove said red blood cell population; and removing said microspheres with said red blood cells bound thereto from said whole blood sample. 7. The method according to claim 6 further characterized by providing magnetic microspheres and a magnetic field and removing said microspheres by removing said red blood cells while attracting said magnetic microspheres within said magnetic field. 8. The method according to claim 6 further characterized by rapidly mixing said microspheres with said whole blood sample to bind said red blood cell population to said microspheres in less than sixty seconds and to provide said lysing action. 9. The method according to claim 2 further characterized by removing said red blood cell population includes providing a red blood cell lyse to substantially eliminate said red blood cell population. 10. The method according to claim 1 further characterized by providing magnetic microspheres and a magnetic field and removing said microspheres by removing said neutrophil populations while attracting said magnetic microspheres within said magnetic field. 11. The method according to claim 1 further characterized by rapidly mixing said microspheres with said sample to bind said neutrophil populations to said microspheres in less than sixty seconds. 12. An apparatus for obtaining a not otherwise obtainable multi-part white blood cell population differential from at least a portion of a whole blood sample having at least white blood cell populations therein for identification and/or enumeration and/or study, characterized by: means for electronically counting at least the total white blood cell populations and the lymphocyte population; means for removing the neutrophil population including the aged neutrophil population contribution from said white blood cell populations by providing microspheres having a neutrophil specific monoclonal antibody bonded thereto and mixing said microspheres with said sample to bind to said neutrophil populations and removing said microspheres with said neutrophil populations bound thereto from said sample and in so doing allowing for the electronic sensing of eosinophils and basophils; means for electronically counting at least said remaining white blood cell populations of monocytes, lymphocytes, eosinophils, aged eosinophils and basophils; and means for comparing said two counts to derive a count of said white blood cell population of neutrophils and aged neutrophils and thereby obtaining at least a five-part white blood cell differential. 13. The apparatus according to claim 12 further characterized by said whole blood sample includes a red blood cell population and means for removing said red blood cell population from said sample without adversely affecting relevant qualities and/or quantities of said white blood cell populations. 14. The apparatus according to claim 13 further characterized by removal of said red blood cell population includes providing microspheres having a red blood cell specific monoclonal antibody bonded thereto and means for mixing said microspheres with said whole blood sample to bind to said red blood cell population; and means for removing said microspheres with said bound red blood cells from said whole blood sample. 15. The apparatus according to claim 14 further characterized by means for providing magnetic microspheres and a magnetic field and removing said microspheres by removing said red blood cells while attracting said magnetic microspheres within said magnetic field. 16. The apparatus according to claim 14 further characterized by means for rapidly mixing said microspheres with said whole blood sample to bind said red blood cell population to said microspheres in less than sixty seconds. 17. The apparatus according to claim 13 further characterized by said means for removing said red blood cell population include providing microspheres having a red blood cell specific monoclonal antibody bonded thereto and means for mixing said microspheres with said whole blood sample to bind to said red blood cell population and providing a red blood cell lyse with said microspheres to eliminate a portion of said red blood cell populations to decrease the number of microspheres necessary to remove said red blood cell populations; and means for removing said microspheres with said red blood cells bound thereto from said whole blood sample. 18. The apparatus according to claim 17 further characterized by means for providing magnetic microspheres and a magnetic field and removing said microspheres by removing said red blood cells while attracting said magnetic microspheres within said magnetic field. 19. The apparatus according to claim 17 further characterized by means for rapidly mixing said microspheres with said whole blood sample to bind said red blood cell population to said microspheres in less than sixty seconds and to provide said lysing action. 20. The apparatus according to claim 13 further characterized by said means for removing said red blood cell population includes providing a red blood cell lyse to substantially eliminate said red blood cell population. 21. The apparatus according to claim 12 further characterized by means for providing magnetic microspheres and a magnetic field and removing said microspheres by removing said neutrophil populations while attracting said magnetic microspheres within said magnetic field. 22. The apparatus according to claim 12 further characterized by means for rapidly mixing said microspheres with said sample to bind said neutrophil populations to said microspheres in less than sixty seconds. 23. A method of obtaining a not otherwise obtainable multi-part white blood cell population differential from at least a portion of a whole blood sample having at least white blood cell populations therein for identification and/or enumeration and/or study, characterized by: electronically counting at least the total white blood cell populations and the lymphocyte population; removing the neutrophil population including the aged neutrophil population contribution from said white blood cell populations from a second portion of said sample without adversely affecting the relevant qualities and/or quantities of said remaining white blood cell populations by providing microspheres having a neutrophil specific monoclonal antibody bonded thereto and mixing said microspheres with said sample to bind to said neutrophil populations and removing said microspheres with said neutrophil populations bound thereto from said sample and in so doing allowing for the electronic sensing of eosinophils and basophils; electronically counting at least said remaining white blood cell populations of monocytes, lymphocytes, eosinophils, aged eosinophils and basophils in said second portion; and comparing said two counts from said first and second portions to derive a count of said white blood cell populations of neutrophils and aged neutrophils and thereby obtaining at least a five-part white blood cell differential. 24. The method according to claim 23 further characterized by said whole blood sample includes a red blood cell population and removing said red blood cell population from said sample without adversely affecting relevant qualities and/or quantities of said white blood cell populations prior to the counting of said first portion and also removing said red blood cell population from said second portion prior to the counting thereof. 25. The method according to claim 24 further characterized by removing said red blood cell population from at least one of said portions includes providing microspheres having a red blood cell specific monoclonal antibody bonded thereto and mixing said microspheres with said whole blood sample to bind to said red blood cell population; and removing said microspheres with said red blood cells bound thereto from said whole blood sample. 26. The method according to claim 25 further characterized by providing magnetic microspheres and a magnetic field and removing said microspheres by removing said red blood cells while attracting said magnetic microspheres within said magnetic field. 27. The method according to claim 25 further characterized by rapidly mixing said microspheres with said whole blood sample to bind to said red blood cell population to said microspheres in less than sixty seconds. 28. The method according to claim 24 further characterized by removing said red blood cell population from at least one of said portions includes providing microspheres having a red blood cell specific monoclonal antibody bonded thereto and mixing said microspheres with said whole blood sample to bind to said red blood cell population and providing a red blood cell lyse with said microspheres to eliminate a portion of said red blood cell populations to decrease the number of microspheres necessary to remove said red blood cell populations; and removing said microspheres with said red blood cells bound thereto from said whole blood sample. 29. The method according to claim 28 further characterized by providing magnetic microspheres and a magnetic field and removing said microspheres by removing said red blood cells while attracting said magnetic microspheres within said magnetic field. 30. The method according to claim 28 further characterized by rapidly mixing said microspheres with said whole blood sample portion to bind said red blood cell population to said microspheres in less than sixty seconds and to provide said lysing action. 31. The method according to claim 24 further characterized by removing said red blood cell population from at least one of said portions includes providing a red blood cell lyse to substantially eliminate said red blood cell population. 32. The method according to claim 23 further characterized by providing magnetic microspheres and a magnetic field and removing said microspheres by removing said neutrophil populations while attracting said magnetic microspheres within said magnetic field. 33. The method according to claim 23 further characterized by rapidly mixing said microspheres with said whole blood sample to bind said neutrophil populations to said microspheres in less than sixty seconds. 34. An apparatus for obtaining a not otherwise obtainable multi-part white blood cell population differential from at least a portion of a whole blood sample having at least white blood cell populations therein for identification and/or enumeration and/or study, characterized by: means for electronically counting at least the total white blood cell populations and the lymphocyte population in a first portion of said sample; means for removing the neutrophil population including the aged neutrophil population contribution from said white blood cell populations from a second portion of said sample without adversely affecting the relevant qualities and/or quantities of said remaining white blood cell populations by providing microspheres having a neutrophil specific monoclonal antibody bonded thereto and mixing said microspheres with said sample to bind to said neutrophil populations and removing said microspheres with said neutrophil populations bound thereto from said sample and in so doing allowing for the electronic sensing of eosinophils and basophils; means for electronically counting at least said remaining white blood cell populations of monocytes, lymphocytes, eosinophils, aged eosinophils and basophils in said second portion; and means for comparing said two counts from said first and second portions to derive a count of said white blood cell populations of neutrophils and aged neutrophils and thereby obtaining at least a five-part white blood cell differential. 35. The apparatus according to claim 34 further characterized by said whole blood sample includes a red blood cell population and including means for removing said red blood cell population from said sample without adversely affecting relevant qualities and/or quantities of said white blood cell populations prior to the counting of said first portion and also removing said red blood cell population from said second portion prior to the counting thereof. 36. The apparatus according to claim 35 further characterized by said means for removing said red blood cell population from at least one of said portions include providing microspheres having a red blood cell specific monoclonal antibody bonded thereto and means for mixing said microspheres with said whole blood sample to bind to said red blood cell population; and means for removing said microspheres with said red blood cells bound thereto from said whole blood sample. 37. The apparatus according to claim 36 further characterized by means for providing magnetic microspheres and a magnetic field and removing said microspheres by removing said red blood cells while attracting said magnetic microspheres within said magnetic field. 38. The apparatus according to claim 36 further characterized by means for rapidly mixing said microspheres with said whole blood sample to bind to said red blood cell population to said microspheres in less than sixty seconds. 39. The apparatus according to claim 35 further characterized by said means for removing said red blood cell population from at least one of said portions includes providing microspheres having a red blood cell specific monoclonal antibody bonded thereto and means for mixing said microspheres with said whole blood sample to bind to said red blood cell population and providing a red blood cell lyse with said microspheres to eliminate a portion of said red blood cell populations to decrease the number of microspheres necessary to remove said red blood cell populations; and means for removing said microspheres with said red blood cells bound thereto from said whole blood sample. 40. The apparatus according to claim 39 further characterized by means for providing magnetic microspheres and a magnetic field and removing said microspheres by removing said red blood cells while attracting said magnetic microspheres within said magnetic field. 41. The apparatus according to claim 39 further characterized by means for rapidly mixing said microspheres with said whole blood sample portion to bind said red blood cell population to said microspheres in less than sixty seconds and to provide said lysing action. 42. The apparatus according to claim 35 further characterized by said means for removing said red blood cell population from at least one of said portions includes providing a red blood cell lyse to substantially eliminate said red blood cell population. 43. The apparatus according to claim 34 further characterized by means for providing magnetic microspheres and a magnetic field and removing said microspheres by removing said neutrophil populations while attracting said magnetic microspheres within said magnetic field. 44. The apparatus according to claim 34 further characterized by means for rapidly mixing said microspheres with said whole blood sample to bind said neutrophil populations to said microspheres in less than sixty seconds. 45. The method as defined in claim 1 wherein comparing said two counts includes comparing the two lymphocyte counts to correlate the population counts. 46. The method as defined in claim 23 wherein comparing said two counts includes comparing the two lymphocyte counts to correlate the population counts. STATEMENTUNDERARTICLE19 Claims 1-48 are cited as stating that the claimed invention cannot be considered to involve an inventive step when Suzuki is combined with one or more other documents, such combination being obvious to a person skilled in the art. The independent claims 1, 12, 23 and 34 (formerly 1, 13, 25 and 37) have been amended to define the specific details relating to neutrophil removal by utilizing microspheres. The amended claims utilizing microspheres clearly are allowable over the reference. Replacement pages 38 - 47 are submitted herewith, incorporating the above amendments. The prior art neither teaches nor suggests the method or apparatus claimed in the amended claims 1-46. The claims present an inventive step over Suzuki. Moreover, no combination of references can be fairly construed to suggest the claimed methods or the claimed apparatus. Accordingly, it is believed that the claims now presented clearly define patentable subject matter over the reference of record.

Inventors: RUSSELL THOMAS, RUSSELL, THOMAS
Applicants: COULTER CORP, COULTER CORPORATION
Application year: 1992
Main classification: G01N33
IPC codes: C12M1, C12Q1, G01N15, G01N33