Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
  • G01N15/12—Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/47—Scattering, i.e. diffuse reflection
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/487—Physical analysis of biological material of liquid biological material
  • G01N33/49—Blood
  • G01N33/491—Blood by separating the blood components
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N2015/1019—Associating Coulter-counter and optical flow cytometer [OFC]
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
  • Y10T436/101666—Particle count or volume standard or control [e.g., platelet count standards, etc.]
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
  • Y10T436/107497—Preparation composition [e.g., lysing or precipitation, etc.]

Definitions

• the present invention relates to a method of analyzing nucleated red blood cells in a blood sample. More specifically the method analyzes blood cell distribution patterns obtained from two separate blood measurement results, determines the presence of nucleated red blood cells, and further enumerates nucleated red blood cells in a blood sample.
• NRBCs Nucleated red blood cells
• erythroblasts are immature red blood cells. They normally occur in the bone marrow but not in peripheral blood.
• a presence of a significant number of NRBCs in peripheral blood may reflect a disturbance of red blood cell maturation, such as megaloblastic anemia, hemolytic conditions including thalassemia, sickle cell crises, leukemia, and transfusion reactions. Therefore, it is of clinical importance to measure NRBCs.
• differentiation and enumeration of NRBC are performed manually. The process involves the smearing of a blood sample on a microscope slide and staining the slide, followed by manual visual analysis of the individual slide.
• the NRBC concentration is reported as numbers of NRBC per 100 white blood cells. Usually, 200 white blood cells and the numbers of NRBC present in the same region on a blood smear are counted and the numbers are divided by 2 to express the NRBC concentration as the numbers of NRBC/100 WBC. This approach is extremely time- consuming as well as being subjective to the interpretation of the individual analyzing the slide.
• U.S. Patent No. 5,298,426 discloses a fluorescence method for differentiating NRBCs.
• the method utilizes a two-step staining using a first fluid and a second fluid.
• the first fluid contains an erythroblast-staining dye that diffuses into nucleated red blood cells to specifically stain their nuclei, and then separating a group of NRBCs from other cell groups on a two-dimensional plot whereby the results of NRBC differentiation are computed.
• the first fluid also contains two additional fluorescence dyes, i.e., an eosinophil/basophil-staining dye and leukocyte-staining dye for specific staining of these cell types.
• two additional fluorescence dyes i.e., an eosinophil/basophil-staining dye and leukocyte-staining dye for specific staining of these cell types.
• U.S. Patent No. 5,559,037 discloses a method for flow cytometric analysis of NRBCs and leukocytes.
• the method comprises lysis of red blood cells and NRBC cytoplasm from a whole blood sample to expose the NRBC nuclei to a vital nuclear stain and minimizing the permeation of the vital nuclear stain into the leukocytes and analyzing the sample by measuring fluorescence and two angles of light scatter.
• This method features a triple triggering method which blocks the signals from debris (fluorescent and non- fluorescent) and identifies the signals which fall below the ALL trigger but above the fluorescence trigger (FL3) as NRBCs.
• ALL is the axial loss of light or the light scatter signals detected at 0° from the incident light. Therefore, pre-gating signals in more than one dimension are required in this method for identification of NRBC population. Since leukocytes are also nucleated cells, staining of these cells needs to be prevented to avoid interference to the fluorescence measurement. The preservation of leukocyte membrane and minimizing the permeation of the nuclear stain into the leukocytes are achieved by concurrently fixing the leukocytes with an aliphatic aldehyde during lysis of red blood cells. In addition, the method requires heating of the reagent to 42°C in order to obtain the NRBC and leukocyte differentiations.
• 5,648,225 discloses a method of using a multipurpose lysing reagent for subclassification of nucleated blood cells.
• the method comprises the steps of lysing a blood sample with the multipurpose lysing reagent which contains a nuclear stain, incubating the sample mixture at an elevated temperature, and determining the nucleated blood cells including NRBCs with an automated electro-optical hematology instrumentation.
• U.S. Patent No. 5,879,900 discloses a method of differentiating NRBCs, damaged white blood cells (WBC), WBC and a WBC differential in a blood sample by flow cytometry.
• the method includes lysing a blood sample; staining NRBCs and any damaged white blood cells with a vital nuclear stain; analyzing the sample mixture by measuring at least one fluorescence, and at least one light scatter signals in a range from 0° to 1° and 3° to 10°; constructing a three-dimensional plot from the fluorescence and light scatter signals; and differentiating and enumerating WBC, NRBC, damaged WBC and a WBC subclass differential.
• EP 1 004 880 A2 discloses reagents and a method for discrimination and counting of nucleated red blood cells. The method includes the steps of lysing red blood cells, staining white blood cells and NRBCs, assaying the sample by measuring at least one scattered light parameter, and at least one fluorescence parameter.
• U.S. Patent No. 5,874,310 discloses a method for differentiation of nucleated red blood cells.
• the method includes lysing mature red blood cells and analyzing the sample in a flow cell by light scatter measurement to differentiate NRBCs from other cell types.
• the light scatter measurement is performed by using two low angle light scatter signals of less than 10°.
• the method further includes a concurrent differentiation of white blood cells using electronic and optical analysis, wherein the electronic analysis is a DC impedance measurement.
• U.S. Patent No. 5,917,584 discloses a method for differentiation of nucleated red blood cells. The method includes lysing mature red blood cells in a blood sample; analyzing the sample in a flow cell by two angles of light scatter measurement to differentiate NRBCs from other cell types, wherein the first light scatter signal is a low angle light scatter signal of less than 10°, and the second light scatter signal is a medium angle or a right- angle light scatter signal.
• WBC white blood cell count
• the present invention relates to a method of analyzing nucleated red blood cells in a blood sample.
• the method comprises the steps of: (a) exposing a first aliquot of a blood sample to a first lysing reagent system to lyse red blood cells and to form first sample mixture, (b) exposing a second aliquot of a blood sample to a second lysing reagent system to lyse red blood cells and to form second sample mixture; (c) measuring the first sample mixture in a flow cell by a detection comprising a direct current impedance measurement (DC1), radio frequency impedance measurement (RF), and light scatter measurement (LS); (d) measuring blood cell distributions of the second sample mixture by a second direct current impedance measurement (DC2) in a non-focused flow aperture; (e) analyzing blood cell distribution patterns obtained from measuring the first sample mixture, and differentiating nucleated red blood cells from other cell types; (f) analyzing blood cell distribution patterns obtained from measuring the second sample mixture, and differentiating
• the method can report the presence of nucleated red blood cells in the blood sample, and can further report the amount of nucleated red blood cells as numbers of nucleated red blood cells per hundred white blood cells in the blood sample.
• the method further comprises counting remaining blood cells in the second sample mixture in the non-focus flow aperture by a third direct current impedance measurement, and reporting numbers of white blood cells in an unit volume of the blood sample.
• the method further comprises reporting an amount of nucleated red blood cells as numbers of nucleated red blood cells in an unit volume of the blood sample.
• Fig. 1A and 1B are the scattergrams obtained from the measurement of the first aliquot of a normal blood sample processed according to the procedure described in Example 1.
• Fig. 1C is a DC2 histogram obtained from the measurement of the second aliquot of the same blood sample.
• Fig. 2A and 2B are the scattergrams obtained from the measurement of the first aliquot of a clinical blood sample containing NRBCs as described in Example 2.
• Fig. 2C is a DC2 histogram obtained from the measurement of the second aliquot of the same clinical sample.
• Fig. 3A and 3B are the scattergrams obtained from the measurement of the first aliquot of a clinical blood sample containing NRBCs as described in Example 3.
• Fig. 3C is a DC2 histogram obtained from the measurement of the second aliquot of the same clinical sample.
• Fig. 4A and 4B are the scattergrams obtained from the measurement of the first aliquot of a clinical blood sample as described in Example 4.
• Fig. 4C is a DC2 histogram obtained from the measurement of the second aliquot of the same clinical sample.
• the present invention is directed to a method for analyzing nucleated red blood cells (NRBCs) in a blood sample.
• NRBCs nucleated red blood cells
• the method comprises the steps of (a) exposing a first aliquot of a blood sample to a first lysing reagent system to lyse red blood cells and to form a first sample mixture, (b) exposing a second aliquot of a blood sample to a second lysing reagent system to lyse red blood cells and to form a second sample mixture, (c) measuring the first sample mixture in a flow cell by direct current impedance measurement (DC1), radio frequency impedance measurement (RF), and light scatter measurement (LS), (d) measuring the second sample mixture by a second direct current impedance measurement (DC2) in a non- focused flow aperture, (e) analyzing blood cell distribution patterns obtained from measuring the first sample mixture, and differentiating nucleated red blood cells from other cell types, (f) analyzing blood cell distribution patterns obtained from measuring the second sample mixture, and differentiating nucleated red blood cells from other cell types, (g) performing a combined analysis on a profile obtained by combining results of analysis from step (e)
• the first lysing reagent system comprises a lytic reagent and a stabilizing reagent.
• a suitable example of the first lysing reagent system is a commercial product, SCATTER PAK ® , manufactured by Beckman Coulter, Inc., Miami, Florida.
• This lysing reagent system has a hypotonic acidic lytic reagent, ErythrolyseTM II, and a hypertonic alkaline stabilizing reagent, StabiLyseTM.
• the composition of the lysing reagent system and method of use for blood analysis are fully described in U.S. Patent No. 5,155,044 (to Ledis et al.), which is hereby incorporated by reference in its entirety.
• the method includes first mixing the first aliquot of the blood sample with a predetermined volume of the lytic reagent to lyse red blood cells, and subsequently adding the stabilizing reagent to retard further lytic reaction in the sample mixture.
• white blood cells in the sample mixture remain essentially intact, and can be used for a subsequent differential analysis. Nucleated red blood cells are more fragile, and their cell membranes are lysed under the same lysing condition. The NRBCs in the first sample mixture have much smaller volume than white blood cells.
• the measurement of the first sample mixture is performed in a focused flow cell using direct current impedance (DC1), radio frequency impedance (RF), and light scatter measurements.
• DC1 direct current impedance
• RF radio frequency impedance
• light scatter measurements When a particle, such as a blood cell, passes through the aperture of a flow cell, an electrical signal can be measured due to conductivity or impedance change.
• the electrical pulse shape, height and width is directly related to the size of a particle or a blood cell, and can be converted to the size of the particles measured.
• the blood cell also scatters the incident light from a laser beam in all directions.
• the light scatter signals can be detected by a light detector at various angles relative to the incident light beam between 0° to 180°. It has been found that each cell population has different light scattering properties, either significant or minor, which might be utilized for differentiation of different cell populations. For the purpose of the present invention, light scatter signals between 10° and 70° are detected for the light scatter measurement. The light scatter signals in this range is also called
• the DC1 , RF and light scatter measurements made on the first sample mixture can produce a series of two dimensional scattergrams using the measured parameters or derivatives of the direct measurements.
• Fig. 1A and 1 B are scattergrams of DC1 versus a first transformed light scatter (RLS), and DC1 versus Opacity (OP, a function of DC1 and RF), respectively, obtained from a normal blood sample processed and analyzed following the procedure described in Example 1. As shown, white blood cells are separated into their subpopulations in the two scattergrams, including lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Importantly, there is no blood cell population in the region below lymphocytes.
• Fig. 2A and 2B are two scattergrams in the same dimensions to those in Fig. 1A and 1B, respectively, obtained under the same condition from a clinical sample that has 5 NRBCs/100 WBC.
• the second lysing reagent system comprises a blood diluent, and a second lytic reagent.
• Suitable examples of the blood diluent are commercial products, ISOTON ® III and ISOTON ® 4.
• Suitable examples of the second lytic reagent are commercial products, LYSE S ® III Diff and LYSE S ® 4. These reagents are manufactured by Beckman Coulter, Inc. Miami, Florida.
• the second lysing reagent system and method of use for blood analysis are fully described in U.S. Patent No. 4,528,274 (to Carter et al.) and U.S. Patent No.
• the method includes first mixing the second aliquot of the blood sample with a predetermined volume of the blood diluent, and subsequently adding a predetermined volume of the lytic reagent to lyse red blood cells.
• remaining blood cells are essentially nucleated blood cells including white blood cells, and nucleated red blood cells if present in a clinical sample. The cell membranes of these nucleated blood cells are damaged by the lytic reaction, which result in substantially reduced cell volumes.
• the population distribution of the nucleated blood cells is measured by a second direct current impedance (DC2) measurement in a non-focused flow aperture to obtain an one dimensional histogram.
• the white blood cells can also be counted in the non-focused flow aperture by a third direct current impedance measurement.
• the detection methods used for blood cell counting by a blood analyzer equipped with a DC impedance measurement device are generally described in U.S. Patent No. 2,656,508 (to Wallace H. Coulter), which is hereby incorporated by reference in its entirety.
• a differential analysis of the DC2 histogram can provide subpopulations of white blood cells.
• Fig. 1C shows a histogram obtained from a normal blood sample processed and analyzed following the procedure described in Example 1. As shown, the white blood cells are differentiated into three subpopulations, importantly, no blood cell population appears on the left of lymphocytes.
• Fig. 2B shows a histogram obtained under the same condition from a clinical sample that has 5 NRBCs/100 WBC. It is apparent that NRBCs appear on the left of lymphocytes on the DC2 histogram.
• a first data analysis for analyzing NRBCs was performed on the blood cell distribution patterns obtained from the measurements of the first sample mixture.
• the analysis includes pattern recognitions of the blood cells in an individual measurement, and derivatives of the measurements, such as DC1 , RF, Opacity (a function of RF and DC1), LS, RLS (a first transformed light scatter), and FLS (a second transformed light scatter). It also includes pattern recognitions of the blood cells in various combinations of two of the measurements and derivatives of the measurements, such as DC1 versus Opacity, DC1 versus RLS, and Opacity versus RLS.
• the analysis further includes a pattern recognition of the blood cells in a combination of the three measurements and derivatives of the measurements, such as a combination of DC1 , Opacity and RLS, and a combination of DC1 , Opacity and FLS.
• the purpose of using derivatives, such as Opacity, RLS and FLS, is to improve population separation among blood cell subpopulations.
• the Opacity is defined as (RF - 85) x 255/DC.
• the RLS is defined as (log 10(LS) - 2.2) x 700000/(DC + 2500) + 50.
• the FLS is defined as (log (LS + 10) - 2.5)/(DC+2500) 1 2 x 4480 - 70.
• the pattern recognition analysis produces numerous variables related to blood cell distribution patterns pertinent to the analysis of NRBCs.
• the variables include percentage of lymphocytes, mean channel of lymphocytes in DC1 , Opacity and RLS, standard deviation of lymphocytes in DC1 , Opacity and RLS, standard deviation of neutrophils in DC1 , percentage of the cell debris cluster, mean channel of the cell debris cluster in DC1 , Opacity and RLS, standard deviation of the cell debris cluster in DC1 , Opacity and RLS, channel of the valley between the cell debris cluster and the white blood cells in DC1 , ratio of the amplitude of the cell debris cluster to the amplitude of the valley between the cell debris cluster and the white blood cells, indication of the blood sample beyond 24 hours, and indication of the presence of a second lymphocyte population (described hereafter). Additional variables can also be generated in the first data analysis to facilitate differentiation of NRBCs from other cell types.
• a database is accumulated which comprises blood cell distribution statistics of the above described variables.
• the first data analyses further differentiate the NRBCs from other cell types, particularly from lymphocytes, cell debris if present, and other cell populations which may appear in the same regions where NRBCs appear.
• NRBCs are more closely positioned to lymphocytes in both DC1 and RLS axes than any other white cell subpopulations. It has been observed that for some abnormal blood samples, a group of lymphocytes having smaller cell sizes appear as a cluster under the normal lymphocyte population in DC1. This cluster of lymphocytes is also called low volume lymphocytes, or second lymphocyte population, for the purpose of the first data analysis. It is also known that lymphocytes of some patient samples, such as patients under chemotherapy, are more fragile, and they tend to overlyse and generate a cluster of partially lysed lymphocyte population, which also appear under the normal lymphocyte population in DC1. In both situations, the first data analysis analyzes the distribution patterns of both lymphocytes and
• NRBCs differentiates the second lymphocyte population from the NRBCs.
• a second data analysis for analyzing NRBCs was performed on the blood cell distribution obtained from the measurement of the second sample mixture. The analysis includes pattern recognitions of the blood cell distribution in DC2 histogram, which produces variables related to blood cell distribution patterns pertinent to the analysis of NRBCs.
• the variables include the channels of the first peak and first valley in DC2 histogram, percentages of lymphocytes, and cell debris in DC2 histogram, the channel of lymphocyte peak in DC2 histogram, the channel and amplitude of the valley between cell debris and lymphocytes in DC2 histogram, ratio of the lymphocyte peak amplitude to the amplitude of the valley between lymphocytes and cell debris, amplitude of the first channel in DC2 histogram, the ratio of the amplitude of the first peak in DC2 to the amplitude of the first channel in DC2, and the separation between lymphocytes and cell debris in DC2 histogram. Additional variables can also be generated in the second data analysis to facilitate differentiation of NRBCs from other cell types.
• a second database is accumulated which includes blood cell distribution statistics of the DC2 histogram.
• the second data analysis also further differentiates the NRBCs from other cell types, particularly lymphocytes.
• DC1 and DC2 have substantially different scales.
• lymphocytes in the first sample mixture are intact, and in their near native states.
• membranes of lymphocytes are lysed, or partially lysed.
• the NRBCs are closer to lymphocytes in DC2 than DC1. Therefore, it is important to differentiate NRBCs from the lymphocytes on the DC2 histogram.
• the method of the present invention performs a combined analysis.
• the combined analysis combines the analysis results obtained from the first and the second data analysis, and generates a profile for the blood sample under analysis.
• the profile includes all above-described variables generated in the first and second data analysis, which contains composite cell population distribution patterns pertinent to NRBC analysis.
• a combined database is accumulated from a large number of normal and clinical abnormal blood samples, which contains various cell distribution statistical information.
• the combined analysis further performs a pattern recognition analysis on the profile of the blood sample. Based on the blood cell distribution statistics the combined analysis then further differentiates NRBCs from other cell types, and obtains the amount of NRBCs present in the blood sample.
• first and second data analysis can face interferences from cell debris, or other cell types such as aged blood cells, platelet clumps and giant platelet, if their sizes coincide with NRBCs in the sample mixtures under analysis.
• NRBCs have a wide size distribution depending on maturity of the cells, and the lymphocytes can also have a wider distribution in abnormal blood samples. All above described situations can complicate one or the other individual data analysis, and cause errors in the analysis of NRBCs if the method is based on only one of them.
• the combined analysis overcomes the deficiencies of individual analysis, and enables a quantitative analysis of NRBC population in the blood sample.
• the method of the present invention takes advantages of the differences by combining the two individual data analysis. It has been found that the cell debris present in the first sample mixture related to the response of the blood cells to the first lysing reagent system often not present in the second sample mixture. In this situation, the interference present from the first data analysis can be removed in the combined analysis. On the other hand, the interference of aged blood cells, platelet clumps and giant platelet to the differentiation of NRBCs in the second sample mixture based on the DC2 histogram may not cause interference in the first data analysis. Hence, the interference can be removed in the combined analysis.
• Example 3 and 4 illustrate the function of the combined analysis.
• the combined analysis reduced the uncertainties or interferences present in the individual analyses, and provided true positive NRBC flags, and the concentrations of NRBCs consistent with the manual reference method.
• the analysis result of the instant method can be reported as a flagging of the presence of NRBCs in a blood sample, and as a concentration of NRBCs in the blood sample.
• the concentration of NRBCs can be reported as number of NRBCs per hundred of white blood cells (NRBC/100 WBC), which is the same unit as the manual reference method, or as numbers of NRBCs in per unit volume of a blood sample.
• NRBC/100 WBC white blood cells
• NRBCs present in a blood sample can elevate white blood cell count, and cause erroneous white blood cell count results.
• the interference of NRBCs can be corrected from the white blood cell count.
• EDTA-anticoagulated fresh normal whole blood sample was aspirated and mixed with a volume of Erythrolyse II in a mixing chamber to lyse the red blood cells, and subsequently mixed with a volume of StabiLyse to retard further lytic reaction in this first sample mixture.
• the first sample mixture was delivered to a focused flow cell with a sheath fluid, ISOTON ® III diluent.
• the ErythrolyseTM II, StabiLyseTM, and ISOTON ® III diluent are products of Beckman Coulter, Inc., Miami, Florida.
• the first sample mixture was measured in the focused flow cell by a detector which included a first direct current impedance (DC1) measurement means, a radio frequency impedance (RF) measurement means, and a light scatter measurement (LS) means.
• the light scatter measurement means detects median angle light scatter signals from about 10° to about 70°.
• a second aliquot of the same blood sample was aspirated and diluted with ISOTON ® III diluent in a WBC bath, and subsequently mixed with a second lytic reagent, LYSE S ® III Diff (a product of Beckman Coulter, Inc., Miami, Florida.)
• the second sample mixture was induced by vacuum to a non-focused aperture embedded in the WBC bath.
• a second direct current impedance measurement (DC2) was performed in the non-focused aperture to obtain a blood cell distribution of nucleated blood cells.
• the white blood cells in the second sample mixture were enumerated in the non-focused aperture by a third direct current impedance measurement.
• the white blood cell count (WBC) was reported as numbers of white blood cells in per unit volume of the blood sample. All sample processes and measurements described above were performed following the instrument operation manual.
• Fig. 1A shows a two-dimensional projection (scattergram) of the three-dimensional scatter plot.
• the vertical axis is DC1
• the horizontal axis is RLS
• a first transformed LS which is a function of DC1 and LS.
• Fig. 1 B shows another two-dimensional projection of the three-dimensional scatter plot.
• the vertical axis is DC1
• the horizontal axis is Opacity (OP), which is a function of DC1 and RF.
• Fig. 1A and 1B illustrate differentiation of white blood cell subpopulations including lymphocytes, monocytes, neutrophils, eosinophils, and basophils.
• a clinical whole blood sample containing 5 NRBCs/100 WBC was processed using the same reagents and procedure described in Example 1 , and measured on a COULTER ® GEN»S instrument under the same conditions.
• the NRBC concentration was obtained from a 100 cell manual count provided by the hospital.
• Fig. 2A and 2B are the obtained DC1 versus RLS, and DC1 versus Opacity scattergram. As shown, the NRBCs appeared in the region below the lymphocytes. Fig. 2B is the obtained DC2 histogram, and the NRBCs appeared in the region on the left of lymphocytes.
• FIG. 3A and 3B are the obtained DC1 versus RLS, and DC1 versus Opacity scattergrams, respectively. As shown, there was a second lymphocyte cluster underneath the normal lymphocyte in DC1 , and NRBCs appeared in the region below the second lymphocyte cluster.
• Fig. 3C is the obtained DC2 histogram. In this case, the lymphocyte population extended into NRBC region, therefore, there was no separation between lymphocytes and NRBCs in the DC2 histogram.
• both the first and the second data analysis indicated the presence of NRBCs.
• the second data analysis was not able to enumerate the NRBCs because of lacking separation between the lymphocytes and NRBCs.
• the first data analysis was able to differentiate the NRBCs from other cell types.
• the combined analysis reported 13 NRBCs/100 WBC, which was consistent with the manual reference report.
• Fig. 4A and 4B are the obtained DC1 versus RLS, and DC1 versus Opacity scattergrams, respectively.
• Fig. 4C is the obtained DC2 histogram.
• the second data analysis was not determinative of the presence of NRBCs.
• Fig. 4A there was a dense cell debris cluster which had a very low DC1 value and small standard deviation in DC1.

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