JP3822625B2 - Hematology control products - Google Patents

Description

  The present invention relates to a novel method for increasing the quality control capability of a hematological instrument system. This method uses (1) D. C. It has particular utility for instruments that measure volume measured by current, (2) high frequency (RF) magnitude, (3) opacity, and (4) light scattering. This method is described in (1) D.D. C. We have found additional utility for instruments that measure volume measured by current and (2) high frequency (RF) magnitude. This method is also described in D.W. C. The utility about the instrument which measures a cell volume only with an electric current was discovered.

  Quality control has long been a necessary and routine technique in clinical hematology. The accuracy in red blood cell and white blood cell counts, including differentiating white blood cell subpopulations, depends in part on the appropriate control product and method of using the control product. In many types of devices for particle counting currently available, there is always a potential for instrument dysfunction, so quality control using control products is required. The traditional method of maintaining a quality control program for automatic particle counters consisted of preparing fresh human blood as a whole blood standard. However, this fresh blood can only be used for one day and therefore a durable blood product has been developed.

  A hematology control product containing a control blood cell analog and monitoring the accuracy and precision of the blood cell counter is important. When using such a blood cell counter, it is recognized that there is a need for new methods that use blood cell analogs to maintain leukocyte differentiation and other parameter accuracy.

  Control products should be as close as possible to that of fresh whole blood. Attempts have been made to prepare stable suspensions of appropriately sized particles using ragweed pollen, polystyrene, latex, various organic materials and immobilized human erythrocytes. None of these suspensions proved suitable for use as a control product for the differentiation of leukocytes of at least four subpopulations of leukocytes.

  Substances used to maintain quality control (hereinafter referred to as hematology control products or control products) can also be used to calibrate hematology instruments under certain conditions. For the purposes of the present invention, the control product used to determine whether the instrument is functioning properly contains one or more analogs suspended in a liquid medium, The analog, when analyzed, simulates at least one physical or biological property of blood that the instrument can analyze. As used herein, an analog is defined as a particle that simulates at least one physical or biological property of a target population. As such, some automatic machines can analyze only certain components of the control product, even though the control product has additional parameter components that are sensitive to analysis by other machines. Traditionally, methods have not yet been developed to use control products to provide quality control of instrument operation. Control products typically provide tests for at least four subgroups of leukocytes, namely lymphocytes, monocytes, neutrophils and eosinophils. Prior art use of control products has been focused on testing whether the instrument provides an appropriate count and percentage of analog. However, the method of the present invention provides additional information for evaluating and diagnosing instrument operation.

  It is clear that the control product must show exactly what the fresh blood test sample constituted for the determination of the problem, based on comparative criteria. In addition, blood components such as red blood cells may slowly lyse after removal from the blood donor and change size and shape within hours, so the control product can simulate fresh blood, It is clear how important it is. Similarly, unstabilized leukocytes suffer from degenerative changes.

  In general, prior art methods of producing analogs have focused on maintaining the original volume prior to fixation or using reduced red blood cells. Contracting or expanding cells by manipulating the osmotic environment prior to fixation had limitations. Traditionally, contracting or swelling non-human erythrocytes by more than about 30% -50% caused excessive cell association or lysis.

  U.S. Pat. No. 3,873,467 (Hunt) describes a blood comprising a washed and stabilized suspension of human erythrocytes in a non-protein aqueous suspension that displaces plasma in human blood. Teaches the clinical control. Stability in the control control includes the cells in an aqueous suspension fluid or substance that allows the cells to take a spherical shape without substantially changing the average cell volume of the cells, as well as resistance to the normal tendency of degradation over time. It is achieved by conditioning the cells by applying the cells. In addition, aqueous suspension fluids create an environment for cells that inhibits biological activity. In a preferred embodiment, a small amount of human red blood cells treated and fixed to have a substantially increased average cell volume is included in the control control. Fixed cells are resistant to changes in cell volume and are resistant to lysis under the action of lysis reagents that lyse stabilized cells. Fixed erythrocytes in the control control replace the leukocyte population in human blood.

  In US Pat. No. 4,704,364 (Carver, et al.), Limitations and additional operational performance for an electron particle counter represented by a COULTER COUNTER ™ S-plus analyzer. Controls are disclosed. However, there is a need for a new method of using a whole blood cell control product for a COULTER ™ VCS analyzer electro-optical particle counter. The VCS analyzer allows discrimination of at least four populations of white blood cells.

  Of automated leukocyte counting of human leukocytes that distinguishes at least four populations of leukocytes in blood from other cells based on size range, volume distribution, light scattering range, electrical opacity and conductivity sensitivity The system requires the control product to accurately simulate the range, distribution and sensitivity of each cell in normal human blood.

  Human lymphocytes, monocytes, neutrophils, basophils and eosinophils have a specific size distribution range and optical properties. Both the upper and lower size limits for each subpopulation of leukocytes should be represented in the control control product. Furthermore, the average cell volume of each leukocyte subpopulation in the control product should approximate that of human blood. Moreover, the liquid suspension medium used in the control product should not cause significant shrinkage or swelling of the cells. Still further, aging of the control product should not degrade the characteristics of the volume distribution histogram or other parameters. A further requirement for leukocyte analogs in control products for multi-parameter instruments is that the analog cells in whole blood control products must not be completely lysed by lysis reagents in order to be counted and distinguished. .

  Various media have been used in combination with blood cell analogs. In U.S. Pat. No. 4,299,726, multipurpose diluents and media are disclosed. The diluent is used to precondition erythrocytes and consists essentially of lactose, sodium azide and a nonionic surfactant, with pH adjusted and osmotic concentration adjusted. The medium is used as a carrier for whole blood control products and includes lactose, fungicides and antibiotics. The medium also contains additional components that alter the membrane of red blood cells, including bile salts and cholic acid derivatives, phenothiazine compounds and their salts with antihistamine properties, and 4-amino-benzoic acid ester derivatives with local anesthetic properties And salts thereof.

  One drawback of the prior art media is that when used in combination with red blood cells and fixed human white blood cells or leukocyte analogs, the control product does not simulate white blood cells in an instrument that distinguishes at least four populations of white blood cells. The specific parameters of the red blood cells and white blood cells to be measured dictate some of the necessary properties of a suitable medium for the white blood cell control control product. It is desirable to know the value of red blood cells. Once this measurement is recognized and red blood cells are counted, the packaged cell volume or hematocrit can be calculated. Therefore, the suspension medium of the control product should be able to equilibrate and stabilize the volume of red blood cells in the sample so that the mean cell volume (MCV) can be measured.

  Also, the control product should be free of particulate matter that presumably interferes with the lower limit of size corresponding to the size and distribution size of human platelets. Concomitantly, the suspension medium optionally includes a bacteriostatic agent to prevent microbial growth after packaging of the control product. Red blood cells and white blood cells usually have different sizes, but their sizes tend to overlap or at least may overlap in some health conditions. Moreover, the opacity of these two types of blood cells can also overlap. Red blood cells and lymphoid leukocytes, unfortunately, have substantial overlap in cell size, and it is not practical to count one in the presence of the other by size discrimination alone. In traditional practice, strong lysis reagents are used, which lyse erythrocytes and make erythrocytes very small particles or solubilize membranes so that red blood cells are not counted, And most if not all of the cytoplasm is removed from the leukocytes, leaving only their lysis-resistant nuclei for counting. Since the cell volume of the original leukocytes is significantly affected and decreases to a minimum, this old form of blood cell size analysis can identify only a single leukocyte population.

  US Pat. No. 3,741,875 (Ansley et al.) Describes a differential leukocyte counting method. A cytological fixative, which is a monoaldehyde, for example formaldehyde, is added to the blood sample. A hemolytic agent is added after the fixation step so that the red blood cells release the hemoglobin content into the solution. The addition of specific cytochemical substrates, chromogenic precipitation coupling reagents, and pH buffering agents precipitate insoluble dyes in specific cell types that contain immobilized enzymes. The solution containing the stained blood cells is then passed through a photometric counter. Using different specific substrates for different enzymes contained in a specific type of cell gives absolute and relative counts of different types of cells. Only monoaldehyde was used as the cytological fixing solution. Dialdehydes are said to be unsuitable because they crosslink and produce extracellular precipitates.

  U.S. Pat. No. 4,485,175 (Ledis, et al.) Uses a quaternary ammonium salt as a lysing agent and a Coulter counter S-plus type automated blood counter (this instrument uses only DC electric field excitation). ) For the method and reagent system for three volume discrimination of leukocyte lymphocyte, mononuclear and granulocyte populations.

  U.S. Pat. No. 4,751,179 (Ledis, et al.) Describes a reagent system comprising a saponin in a rapidly active cross-linking agent, e.g. glutaraldehyde, as a lysis reagent and fixative, said system comprising Reproducibly affects whole blood to lyse red blood cells and denature white blood cells to generate data that reveals four distinct clusters for detection and classification by flow analysis instruments. Clusters represent the four major leukocyte types found in blood, namely lymphocytes, monocytes, neutrophils and eosinophils, thus providing a differential analysis of leukocytes. Ledis, et al. According to D. C. Traditional methods of leukocyte flow analysis using light scatter at volume or at different angles showed only two clusters of leukocytes, corresponding to lymphocytes, granulocytes and monocytes. For classification of leukocytes, Ledis, et al. The parameters used by C. (Coulter) volume, high frequency (RF) magnitude, Coulter opacity (RF magnitude / DC volume), light scattering in different angular ranges, and fluorescence at different illumination wavelengths Includes more combinations.

  An electronic counter using the Coulter principle was first described in US Pat. No. 2,656,508 and represents a true reflection of particle count. According to the Coulter principle, there is an instantaneous change in the electrical impedance of the electric field when microscopically sized particles are suspended in an electrolyte liquid and passed through a small sized electric field that approaches the size of the particle. Will. When the electric field is excited by direct current (DC) or low frequency current, the electrical change is closely proportional to the value of the particles. In commercial equipment, changes are detected by several appropriate meanings and are used to operate counters and analyzers. An analyzer associated with such a device sorts and sorts particles into a population based on the volume of the particles.

  The Coulter Principle invention was substantially expanded in US Pat. No. 3,502,974 (Coulter, et al.), Where it was studied using radio frequency (RF) current in addition to DC field excitation. In addition to DC volume information on the particles to be collected, information is also obtained on the composition and nature of the materials that make up the particles. This patent discloses a device that can distinguish between particles of the same size but different materials. Inducing two or more output signals from the passage of a single particle through the electric field by generating a particle sensitive electric field by both low frequency or direct current (DC) and radio frequency (RF) current excitation. be able to. This is due to the facts below. That is, particles, eg, cells, are almost always insulated with respect to low frequency or DC electric fields, but particles can carry or block radio frequency currents differently from the surrounding electrolyte. This is due to the difference in dielectric constant in the case of homogeneous particles, or because of the sac-like structure in the case of blood cells with contents that have a different conductivity from the electrolyte, enclosed in a very thin film. It is. Thus, all DC current goes around the blood cell, but some of the RF current passes through it. The ease with which an RF current passes through a particle, like light transmission, is a measure called “electrical transparency” or simply “transparency”, whereas the ability of a particle to interfere with RF current is It is called “transparency”. In later publications, “opacity” is defined as the RF impedance divided by the DC impedance.

  The relative electrical opacity of the particles is characteristic of the identification of the particle content and hence the particle type for classification purposes. To the extent that each of the different types of particles has a different opacity, the difference between the particles can be detected. However, significantly different particles can have substantially the same transparency, and in this way such particles cannot be effectively classified. In US Pat. No. 3,836,849 (Coulter, et al.), It is possible to selectively change the transparency of the particle type by treating the particles so that a detectable result is obtained. Taught.

  The Coulter Counter S-Plus automated blood cell counter is designed to dilute a sample of whole blood in an isotonic diluent, add a lysing agent, and then start counting in a short time. Thus, the diluent-lysate system must provide a erythrocyte lysis reaction rate that is sufficiently rapid to perform complete interstitial lysis of red blood cells during the lysis period. Furthermore, changes in white blood cell volume should be minimal during the data collection step and ideally should be stable for several minutes.

  The Coulter VCS model is a semi-automated analytical instrument that analyzes blood by using DC (Coulter) volume, Coulter transparency, and light scattering in various angular ranges. Coulter VCS uses a reagent system to obtain a five-part distinction in the total white blood cell count and provides a quantitative analysis of a population of lymphocytes, monocytes, neutrophils, eosinophils and basophils To do. The reagent system includes a quench, which is added after weak “acidic” lysis, which is manipulated to greatly reduce the lytic effect on leukocytes. After a short period of quench, the instrument begins to measure residual white blood cell volume, clarity and light scatter characteristics. The VCS type provides erythrocyte lysis kinetics sufficiently rapid to effect complete interstitial lysis of red blood cells during the lysis period, but affects the properties of white blood cell volume, Coulter transparency and light scattering. Must not. Coulter counter devices that can be used in the present invention are VCS, STKS and MAXM. However, types S and S-plus cannot distinguish all of the leukocyte subpopulations present in whole blood control products, but rather can provide a total count of leukocyte analogs. In addition, some of the S-plus types can distinguish between two leukocyte populations.

  The new electro-optic particle counter requires the development of a new method to determine whether the instrument is functioning properly within the manufacturer's specifications and diagnosing the cause of instrument malfunction. This standard is primarily directed to methods of using hematology control products that are useful with Coulter-type particle counters, but are described in the suspension media, analogs and control products disclosed herein, and herein. It should be understood that their usage generally finds wide application with particle counters. Thus, the term “electro-optic particle counter” refers to an electronic identification circuit (“limit”) that responds electronically to signals indicative of particle size, mass, volume, transparency, or light scattering, in addition to a Coulter counter instrument. It should be understood to encompass any other type of particle counter that identifies particles of various sizes. Coulter and Coulter Counter are registered trademarks of Coulter Corporation.

  The present invention relates to a method of using a hematology control product, said method comprising placing the hematology control product in an instrument, said control product comprising at least one leukocyte analog. The leukocyte analog is derived from blood cells that have been treated to be resistant to degradation by the lytic reagents used in hematology testing procedures, and the analog is used in the operation of the instrument. Responsiveness to reagents. A new control parameter is used to analyze the spatial location of the control product. C. Selected from at least one member of the group consisting of volume, RF magnitude, opacity, and light scattering. More preferably, at least two different physical properties are measured. The results of such measurements are then reported to diagnose the cause of instrument dysfunction.

  The present invention further relates to a method of using a control product containing a population of at least one leukocyte analog to determine whether the instrument is functioning within the manufacturer's analytical specifications. The method comprises placing a hematology control product in the instrument, the control product containing at least one leukocyte analog, which is used in a hematology test procedure. Derived from blood cells that have been treated to be resistant to degradation by lytic reagents, and the analogs remain responsive to instrument operation. The control product simulates at least one physical property of human leukocytes, said property being C. Selected from the group consisting of volume measured by current, high frequency (RF) magnitude, opacity, and light scattering. More preferably, the control product simulates at least two physical properties of human leukocytes. Then, at least one, and preferably at least two physical properties are determined from the physical properties of the control product. Report the results of such measurements to diagnose the cause of instrument dysfunction.

  Still further, the present invention analyzes the spatial location of a subpopulation of leukocytes in an amount of a patient's blood sample to obtain a statistically significant value of the measured parameter, said analysis comprising: . C. Selecting from at least one member of the group consisting of volume, RF magnitude, opacity, and light scattering, and reporting the results of said measurement on the instrument to diagnose the cause of instrument dysfunction About how to become.

Moreover, with respect to combining a hematological sample with a cell suspension comprising an aqueous solution of plasma material and analyzing the resulting mixture in an instrument to diagnose the cause of dysfunction, said analysis comprises C. It is selected from at least one member of the group consisting of volume, RF magnitude, transparency, and light scattering, more preferably at least two members.
Detailed Description of the Invention For purposes of understanding and explaining the present invention, the following terms are defined.

  Beads: Beads are particles (usually made from polystyrene latex or some form) that can be used as a stable and inert standard for flow cytometric analysis. Beads can be obtained in different narrow defined sizes to standardize FSC (standard format for flow cytometry data storage) settings. The beads can also be obtained conjugated to various fluorescent dyes to standardize the fluorescence detection settings.

  Channel: Channel is a term used when a flow cytometer characterizes the intensity of a signal emitted by a particle. Most cytometers divide the optical signal into 256 for 1024 channels. A signal with a higher channel number is brighter than a lower channel number. However, the quantitative relationship between a signal defined by one channel number and a signal defined by another channel number will depend on the voltage characteristics of the amplifier and photodetector of a given protocol.

  Coaxial flow: A narrow core flow of liquid in a wider flow. This type of flow is important in flow cytometry. This is because it tightly constrains the particles flowing through a relatively wide nozzle spatially and allows accurate and stable illumination when the particles sequentially pass through the light beam.

  Compensation: Compensation is the ability of a flow cytometer to correct for overlap between the fluorescence spectra of different fluorochromes. Without compensation, the fluorescence from a given fluorescent dye may be recorded to some extent on a photodetector that is assigned to detect different fluorescent dyes.

  Coulter volume: When the particles flow through a narrow nozzle, the increase in electrical resistance that occurs when the particles displace the electrolyte is the Coulter volume of the particles. This increase in electrical resistance is only roughly related to the volume of the particles.

  Core: The core is an inflow that is injected into the center of the sheath flow and maintained there by laminar hydrodynamic considerations. The core contains the sample treatment to be analyzed in a flow cytometer.

  Crosstalk: Crosstalk is a signal from a “false” photodetector that is obtained through a filter on the photodetector where the fluorescence emitted by one fluorochrome is usually specific for fluorescence from a different fluorochrome. This occurs because it contains some light of wavelength. See “Compensation”.

  CV: The coefficient of variation is defined as the standard deviation of a series of values divided by the average of those values. It is used in flow cytometry to describe histogram peaks. In one protocol, it can be used to assess variations in the properties of particles within a population. In DNA analysis (where all normal particles are assumed to have the same properties), it can often be used to assess flow cytometry alignment (and operator skill).

  Fixation: A method of denaturing cellular proteins. Establishment in flow cytometry is used to preserve the stained cells that inactivate dangerous biological material and also lack immediate access to the flow cytometer. Paraformaldehyde is the fixative selected in flow cytometry because it preserves the forward and lateral scattering properties of the cells (but somewhat increases autofluorescence).

  Flow cell: A flow cell is a device in a flow cytometer that delivers a sample flow to the center of the sheath flow. In some cytometric configurations, illumination is performed “in air” after the flow leaves the flow cell.

  Fluorescent dye: A fluorescent dye is a dye that absorbs light and then emits a different color of light (always with a longer wavelength).

  Forward scattering: Forward scattering is light from an illumination light beam that is bent so that the light passes through the particle and diverges from the original direction of the light beam. The intensity of light bent at a small angle from the illumination light beam is related to the refractive index of the particles as well as the cross-sectional area. The forward scatter signal does not correlate with cell volume.

  Gain: Gain is an electronic control to the amplifier that determines the current density that occurs when a photomultiplier receives a given signal. Variations in the gain of photomultiplier tube amplifiers can change the appearance of those signals as the output signals are converted to flow cytometry data.

  Gate: A restriction made on flow cytometry data that is subsequently analyzed. Live gates limit the data that a computer receives for storage. An analysis gate simply excludes certain stored data from a particular analysis technique. Gates are used to limit the analysis of a mixed population to certain types of cells within that mixed population.

  Granularity: Granularity is a term used interchangeably with side scatter to describe light deflected at right angles from the illumination light beam in a flow cytometer. This light intensity is related in an inaccurate manner to the internal or surface irregularities of the particles flowing through the light beam.

  Light Scattering: Median angle light scattering (MALS) is defined as light scattering information obtained at angles between 10 ° and 70 °. Low angular light scattering (LALS) is light scattering information obtained at angles of 10 ° or less with respect to the light beam axis, excluding 0 °. High angular light scattering (HALS) is light scattering information centered at 90 ° to the laser axis.

  Linear amplifier: A linear amplifier is one means by which the signal from a photomultiplier can be increased and measured. Linear amplifiers increase the signal in such a way that the output current from the amplifier is directly proportional to the input current induced from the photodetector.

  Logarithmic amplifier: A logarithmic amplifier is one means by which the signal from a photomultiplier can be changed and measured. The logarithmic amplifier changes the signal in such a way that the output current from the amplifier is proportional to the logarithm of the input current derived from the photodetector.

  Photodetector: A photodetector is a device that senses light and converts the energy from that light into an electrical signal. Within the operating range of the detector, the electrical signal is proportional to the light intensity. Photomultiplier tubes and photodiodes are two types of photodetectors.

  Photodiode: A type of photodetector that is used to detect relatively strong optical signals. It has no high voltage applied to increase the current at its anode (output) end.

  Photomultiplier tube: A photomultiplier tube is a type of photodetector that is used to detect relatively weak signals. Its output current is increased by the applied high voltage.

  Rotating light scattering: Rotating light scattering (RLS) is a transformation of data derived from the information ratio of MALS / DC pulse peaks. The function of RLS has the effect of removing cell size components and producing measurements that are more related to the internal structure of the cell.

  Sheath: A fluid in which a central sample core is contained within a flow cell of a flow cytometer or during coaxial flow therein.

  Side scatter: Side scatter is light of the same color as the illumination light beam that bounces off particles in the illumination light beam and deflects to the side. The “side surface” is usually defined by a lens that is perpendicular (orthogonal) to the light beam line. It is also referred to interchangeably as right-angle light or 90 ° LS. This intensity scattered to the side is in a general way related to the roughness or irregularity of the surface or the interior of the particles.

  Spatial position: Spatial position is the position of the analyzed population in the domain of analysis. In the example of a VCS instrument, the domain of analysis is D.I. C. Current, high frequency (RF) magnitude and volume measured by light scattering. Another example is that the analyzed population is D.P. C. This is the case only by the volume measured by the current, and the spatial position is D.D. C. It will be the average position of the analyzed population within the domain.

  Limit: A limit is an electronic device that can make an ADC to ignore signals below a certain intensity. The forward scatter limit is most commonly used in flow cytometry to exclude very small particles, debris and electronic or optical noise from acquisition.

  VCS technology: An instrument that analyzes blood by using DC (Coulter) volume, Coulter volume or RF magnitude, and light scattering in various angular ranges.

  Wavelength: Wavelength is a characteristic of light that is precisely related to its energy content and also its color (using light to which our eyes are sensitive). Short wavelength light has more energy than longer wavelength light.

  Analysis of current multi-white blood cell populations requires specific size and volume increments and analogs of specific light scattering properties for use as quality controls. In the method of the present invention, at least one analog of a major leukocyte component that is a lymphocyte, monocyte, neutrophil, and eosinophil is used to test the limit setting of the electro-optic particle counter. It is necessary to prepare. Preferably, it is necessary to prepare at least a neutrophil analog for the method of the invention. More preferably, it is necessary to prepare analogs of neutrophils and lymphocytes. Prior to this, the increase in volume was associated with an increase in light scatter, which prevented the creation of at least four different populations of leukocyte analogs other than human leukocytes. Analogs and instruments used to analyze analogs become more complex, and the suspension medium of the analogs must supplement their complexity. More particularly, the suspending medium must be compatible with these analogs and devices and supplement the physical and physiological properties of the analogs.

  Suspension media are primarily used with leukocyte analogs prepared from blood. One method of preparing leukocyte analogs provides processed blood cells from different sources to meet multiple limit settings for multiple types of blood counting instruments. In selecting blood cells, the main limitation is the average cell volume of the original cells. Because it is related to the average cell volume of the desired analog. Without limiting the scope of the invention, it should be understood that blood cells from a particular animal can be specifically described, where red blood cells and white blood cells from other animals can be used in the methods of the invention.

  One method for preparing leukocyte analogs useful in the methods of the present invention is to mix red blood cells with a hypotonic solution to expand the volume of the cells and change the hemoglobin content of the cells to increase the light scattering and clarity of human leukocytes. D. Simulating and fixing the leukocytes, resistant to degradation by the lysis reagent used in the hematology test procedure, and the fixed leukocytes are similar in nature to human leukocytes; C. The current comprises measuring at least two properties selected from the group consisting of measured volume, high frequency (RF) magnitude, transparency and light scattering. Although the process for producing blood cell analogs of eosinophils is similar, changes in hemoglobin content are achieved by denaturing it in cells rather than letting it leak out of blood cells. Additional embodiments result in analogs having the volume and light scattering properties of human leukocytes.

  This method can also cause the red blood cells to swell 50% more than the original volume, which provides greater latitude in the selection of animal cells for the preparation of the desired analog. In a preferred method, red blood cells are swollen more than 75% of their original volume.

  For the purpose of preparing suitable analogs for use with the methods of the present invention, poultry erythrocytes such as turkey, chicken, duck, and preferably cancer erythrocytes are stabilized with aldehydes to produce smaller leukocyte analogs. It was discovered that it can be generated. Also, other non-human vertebrates, including “fish”, instrument movement sharks and reptiles, preferably alligators, when properly treated are larger human monocytes, neutrophils and eosinophils. It has been discovered to have a desired size range of red blood cells that give analogs similar to spheres. These red blood cells generally exhibit very good suspension capacity and highly reproducible volume distribution characteristics. However, consideration must be taken into account, for example the availability at reasonable costs.

  Moreover, when determining white blood cell parameters in a whole blood control product, the red blood cells are fixed so that the red blood cells are resistant to degradation by the lysis reagent used in the hematology test procedure.

  Avian, alligator and guinea pig cells are nucleated, but given the methods described herein that allow for regulated hemolysis of erythrocytes, the use of said cells as an alternative to human leukocytes, The presence of the nucleus is not essential and is not critical. Preferably, 20-80%, more preferably 30-70% by weight of the hemoglobin in the cells is released. Stabilize cells with a fixative that prevents cell membrane disruption and further loss of hemoglobin, such as organic aldehydes.

  Another method of preparing leukocyte analogs useful in the methods of the present invention involves stabilizing human leukocytes to simulate at least one of five subpopulations of leukocytes.

  Furthermore, the methods of the present invention are useful when using other leukocyte analogs known in the art. These stabilized leukocyte analog cells provide a satisfactory alternative for human leukocytes in control products.

  A preferred method of producing a suitable control product for use in the methods of the present invention is to simulate fixed cancer erythrocytes, human monocytes, neutrophils, and eosinophils to simulate human lymphocytes. A composition prepared by mixing a suspension of alligator erythrocytes immobilized on and assembled together in a suspending medium and in a ratio that forms a single composition to simulate human leukocytes To materialize. The control product is then mixed with lysable human erythrocytes and stabilized platelets or platelet analogs to form a single multi-analytical control product.

  The following description illustrates a preferred method for preparing control products using red blood cells for use in the methods of the present invention.

  In the collection step, red blood cells are suspended in an anticoagulant, for example, an alkali metal salt of ethylenediaminetetraacetic acid (EDTA) dissolved in a physiological saline solution (sodium chloride). Other anticoagulants and salts are considered effective unless they cause adverse hemolysis or cell association.

  Fresh red blood cells must be washed to remove donor-specific plasma passage. This reduces the likelihood of red blood cell aggregation when mixing red blood cells from multiple blood cell donors. Red blood cells are pooled together to obtain a homogeneous complex.

  The red blood cell pool can be pretreated with serum material as a processing aid. Pretreatment using serum material allows the red blood cells to swell without rupturing the red blood cells. Exposure of red blood cells to a hypotonic environment has the main effect of increasing the mean cell volume and decreasing the width of the light scatter histogram. As a result of the hypotonic environment having a solute concentration that is reduced from that of red blood cells, the size of red blood cells increases. When the solute concentration inside the red blood cell is greater than the concentration outside the red blood cell, there is a tendency for water to move into the red blood cell and equilibrate the concentration. As such, the movement of water inside the red blood cells causes swelling. The hypotonic environment can include solutions of sodium compounds, potassium compounds or both, or other compositions known to those skilled in the art to provide the desired solute concentration.

As defined herein, serum comprises cholesterol, cholesterol esters, and cholesterol combined with one or more other compounds found in serum plasma, and mixtures thereof. Preferably, such other compounds further comprise lipoproteins and phospholipids and mixtures thereof. As those skilled in the art will appreciate, typically cholesterol will contain approximately 30% esters. Furthermore, as those skilled in the art will appreciate, lipoproteins will maintain cholesterol in aqueous solution. Preferably, the serum substance in the pretreatment is selected from the group consisting of cholesterol, cholesterol esters, lipoprotein cholesterol, lipoprotein cholesterol esters, phospholipid-conjugated cholesterol and mixtures thereof. Most preferably, the serum material comprises cholesterol cholesterol combined with phospholipids. A suitable commercially available example of such a preferred embodiment is Pentex ™ Cholesterol Super-Trate (Miles, Inc.), which is a lipoprotein cholesterol ester in combination with high density lipoprotein cholesterol and phospholipids. . Thus, pretreatment is necessary when smaller blood cells swell more than 30-50% of the original volume. Furthermore, the concentration of serum substance used is believed to be both a function of the amount of blood cell swelling caused by the hypotonic solution, as well as the process conditions of the fixed reaction that cause the hemoglobin of blood cells to leak from the blood cells. Pretreatment seems unnecessary in processes where blood cells are fixed within approximately 2 hours, primarily due to aldehyde concentrations at room temperature, and low osmotic pressure is approximately 150 milliosmolar. When using pretreatment, preferably the cholesterol concentration is 0.1-5.0 mg for a blood count of 1 × 10 ⁶ blood cells / μl. When using cholesterol concentrations that are too high, blood cells tend to lyse. If cholesterol concentrations that are too low are used, blood cells will rupture as they swell.

  Prior art attempts to swell blood cells without rupturing the blood cells have focused on the use of processing aids that function to strengthen the cell membrane, such as potassium sodium tartrate. However, this approach does not allow the expected expansion of 30-50% and does not provide blood cells with controlled hemolysis.

  The method of the present invention is disclosed by simultaneously swelling and fixing blood cells in a one-step process, but using two or more steps, blood cells are pretreated with serum material and blood cells are swollen to control hemoglobin. It is within the scope of this method that it can be released and then the blood cells can be fixed. However, such a technique is expected to have a problem of controlling the conditions for each step, more specifically, the timing of blood cell fixation.

  In a preferred method of producing a control product for use in the method of the present invention, a low osmotic solution is formed by combining an aqueous solution of sodium phosphate with a fixed reagent and the desired osmotic pressure. The lower the osmotic pressure with respect to the normal tonicity of natural blood, the more blood cells will swell due to the water that moves partially from the outside of the blood cell to the inside of the blood cell. The osmotic pressure is preferably 0-150 milliosmolar, even more preferably 65-95 millimolar for eosinophil analogs, depending on the initial blood cell size, blood cell count, and desired final blood cell size. The osmolarity ranges from 0-20 milliosmolar for monocyte analogs, 5-35 milliosmolar for leukocyte analogs, and 45-65 milliosmolar for neutrophil analogs. The preferred range above is based on blood cells washed with isotonic saline, and further based on blood cell counts in a fixation reaction of approximately 20,000-50,000 blood cells / μl.

  Concomitantly, it does not appear to affect the swelling rate of temperature blood cells independently, and does in fact affect the fixed reaction rate. As the blood cells expand, hemoglobin leaks out of the blood cells at a controlled rate until the fixation reaction prevents further release of hemoglobin. Most of the hemoglobin will be released within the first 5 minutes of the hypotonic treatment. Therefore, in the simultaneous swelling and fixation of blood cells, reducing the fixation temperature in the solution can control the fixation process and the release rate of hemoglobin during the blood cell swelling. When the fixation reaction is complete, the blood cells are resistant to lysis or degradation under the influence of normal lysis reagents used in hematological testing procedures.

  In another method of producing a control product for use in the method of the present invention, blood cells are added to a chilled hypotonic solution containing glutaraldehyde. The chilled fixation solution is at a temperature of 0-15 ° C, more preferably 1-10 ° C, and most preferably the fixation process is performed at 2-8 ° C for lymphocyte and monocyte analogs at neutrophils and eosinophils. For analogs of at room temperature. The reduced temperature has been shown to provide quantitatively different blood cells as measured by a sorting device, such as a Coulter Counter VCS analyzer. Quantitative differences include higher average cell volumes compared to fixation at room temperature.

  Fixation of swollen blood cells is important for strengthening the cell membrane and preventing membrane degradation. This is accomplished by contacting the blood cells with a solution of an organic aldehyde, such as a monoaldehyde, such as formaldehyde, or a dialdehyde, such as glutaraldehyde. Glutaraldehyde is a preferred aldehyde because it reacts more rapidly than formaldehyde. As long as the final concentration is in the range of about 0.05% to 0.8%, more preferably 0.1% to 0.6%, based on a blood count of approximately 20,000 to 50,000 blood cells / μl Glutaraldehyde can be added at a concentration higher than the final concentration. Practical limits on the selection of the appropriate aldehyde and its concentration are as parameters in blood cell count, functional limitation of elimination of adverse blood cell associations, and control of fixation reactions. Fixation reaction conditions will vary for the particular animal cells used and the leukocyte analogs prepared.

  Most room temperature fixation using glutaraldehyde occurs within 2 hours, but longer to make the erythrocytes fully resistant to the usual erythrocyte lysing agents used in Coulter counter hematology instruments Time is required. With careful selection of red blood cells, the length of time for fixation using glutaraldehyde can vary from 2 to 72 hours, preferably depending on temperature, glutaraldehyde concentration, blood cell count and the desired amount of hemoglobin released. It will be in the range of 3-30 hours. In a most preferred embodiment, the fixed time for a blood cell count of approximately 20,000-50,000 blood cells / μl is 10-24 hours for monocyte and lymphocyte analogs, and for eosinophils and neutrophils 3-18 hours for analogs. Incomplete fixation may occur in partially fixed red blood cells having a lower average cell volume for the targeted population of human leukocytes. In general, the upper limit of fixation is based on the convenience of preparation. After fixation, blood cells are separated from the liquid phase by centrifugation and gravity means and then washed with phosphate buffered saline.

  The pH of the fixing solution is 7.0-9.0. If the pH of the fixing solution is too low, agglomeration occurs and if it is too high, the blood cells may rupture. Furthermore, pH affects the release of hemoglobin. If the fixation reaction occurs too quickly, the cells cannot release hemoglobin. Accordingly, the pH range is approximately 7.0 to 9.0, preferably 7.5 to 8.5. In the most preferred embodiment, the pH of the fixing solution is 8.0 ± 0.2 for neutrophil and eosinophil analogs and 7.8 ± 0.1 for monocyte and lymphocyte analogs. .

  Eosinophil analogs are prepared in a similar manner, except that hypotonic glutaraldehyde solutions are preferably prepared at room temperature, and hypotonic glutaraldehyde solutions are hemoglobin in blood cells rather than completely fixing the cells. Is mainly used for lightly crosslinking. As such, the concentration of glutaraldehyde for a blood cell count of approximately 20,000-50,000 blood cells / μl is approximately 0.1-0.4%, more preferably 0.2-0.3%. After lightly cross-linking hemoglobin and washing with phosphate buffered saline, the blood cells are protein denaturing reagents, such as quaternary ammonium compounds, or other known to those skilled in the art to precipitate hemoglobin in the blood cells. Further treatment with a denaturant. The pH of the denaturing solution should be 9.0-12.0, preferably 10.0-11.0. Treatment does not reduce blood cell volume. Treatment with a protein denaturing reagent increases the light scattering properties of the swollen blood cells to provide swollen blood cells with the required light scattering properties similar to human eosinophils. Both degeneration of hemoglobin and the controlled release of hemoglobin have the effect of changing the hemoglobin composition in the blood cells. However, the nature of light scattering differs significantly between the controlled release of hemoglobin in monocyte and lymphocyte analogs and the degeneration of hemoglobin in eosinophil analogs. In general, release of hemoglobin from blood cells will reduce blood cell light scattering and transparency. Degeneration of hemoglobin in blood cells will increase blood cell light scattering.

  A preferred method for preparing eosinophil analogs comprises pretreating a pool of red blood cells with aqueous serum material, swelling the red blood cells, denaturing hemoglobin in the red blood cells, and fixing the red blood cells. As those skilled in the art will appreciate, producing a control product that can select the appropriate size of red blood cells that does not require the amount of swelling that requires pretreatment with serum material is an Within range. In such cases, the method denatures hemoglobin in erythrocytes to simulate the light scattering properties of human leukocytes, and is resistant to degradation by lytic reagents used in hematological testing procedures. Immobilizing red blood cells. As such, treated red blood cells will have light scattering and volume properties similar to human leukocytes. However, if the red blood cells do not swell to some extent, the standard deviation of light scatter will not fall within the boundaries of the largest cell population because the red blood cells are not naturally spherical. The addition of a spheronizing agent can eliminate this problem.

  By using a combination of the processing steps disclosed above that swell the red blood cells, release the hemoglobin from the red blood cells, denature the released hemoglobin, and contract the red blood cells by methods known to those skilled in the art. D. can be used in a novel method for diagnosing the cause of dysfunction C. A method is obtained for designing analogs with multiple different physical parameters of volume, RF magnitude, transparency and light scattering. More specifically, erythrocyte contraction and swelling affects all of the above parameters, but changes in hemoglobin in erythrocytes can affect transparency and light scattering properties.

  The suspending medium disclosed herein is also used with leukocyte analogs prepared by other methods known in the art. One such other method involves fixing human leukocytes to simulate five subpopulations of leukocytes as described in Example 5.

  A control blood cell control product can include one or more leukocyte analogs prepared by any of the methods known in the art or any of the foregoing methods. The leukocyte analog can be any suitable medium, such as phosphate buffered saline and US Pat. No. 4,213,876, US Pat. No. 4,299,726, US Pat. No. 4,358,394 and US It can be stored in the medium described in detail in patent 3,873,467.

  The following specific examples are disclosed in US Pat. No. 4,299,726.

Stabilizing medium that confers long-term stability to erythrocytes-preferred formulation Approximate liter formulation 500ml distilled water
2. Propylparaben 0.3-1.0g
3. Methyl paraben 0.5-1.0g
4). Procaine hydrochloride 0.1-0.5g
5). Deoxycholic acid 0.1-0.9g
6). Lactose 10.0-50.0g
7). Actidione 0.1-0.6g
8). Trisodium citrate 2H ₂ O 3.0-8.0 g
9. Citric acid 1H ₂ O 0.3-0.9 g
10. Sodium dihydrogen phosphate 1H ₂ O 0.8-2.5 g
11. Fenergan hydrochloride 0.1-1.0g
12 Colistimate, sodium 0.2-0.9g
13. Penicillin G, sodium 0.5 × 10 ^{6 to} 3 × 10 ⁶ units14. Kanamycin sulfate 0.2-0.8g
15. Neomycin sulfate 0.2-1.0g
16.5'-AMP 0.4-1.0g
17. Adenine 0.2 ~ 0.8g
18. Inosine 0.4-1.0g
19. Dihydrostreptomycin sulfate 0.2-1.0 g
20. Tetracycline hydrochloride 0.2-1.0g
21. 30% bovine albumin 100-350ml
22. Amount to make 1 liter of distilled water

  Since many of the chemicals listed above are commercially known under a number of names, the names given are Merck Index, 7th Edition (1989), Merck and Co. , Inc. It is a common name listed in Raway, New Jersey.

  When manufacturing the control product, the supernatant fluid is removed from the leukocyte analog and the leukocyte analog is then resuspended in the suspending medium. A preferred suspending medium comprises an aqueous solution of plasma material. As defined herein, an aqueous solution of plasma substance comprises an aqueous solution of serum substance (as defined above), a combination of serum substance and plasma protein, and mixtures thereof. As further defined herein, plasma proteins comprise one or more proteins contained in plasma. Preferably, such plasma proteins comprise albumin, lipoprotein, globulin, fibrinogen, and mixtures thereof. More preferably, the plasma substance is cholesterol, cholesterol swell, lipoprotein cholesterol, lipoprotein cholesterol ester, cholesterol bound to phospholipid, cholesterol bound to albumin, cholesterol ester bound to albumin, lipoprotein cholesterol bound to phospholipid, Selected from the group consisting of lipoprotein cholesterol combined with albumin, and mixtures thereof.

In order to confirm the usefulness of plasma material for lysis of red blood cells in a saponin-based lysis system, aqueous plasma material is added to the washed red blood cells. The aqueous solution contained 3% plasma material in phosphate buffered saline. The results are as follows:
Sample Plasma substance Dissolution 1. Human albumin Yes 2. Albumin from which fatty acids have been removed None. Sample 2 with lipoprotein cholesterol Yes 4. Bovine serum albumin None5. Sample 4 with lipoprotein cholesterol Yes 6. 6. Albumin-bound cholesterol Yes. Monomeric albumin Yes8. Bovine serum albumin, caprate stabilization None. Sample 8 with lipoprotein cholesterol Yes10. Polymer enhanced bovine serum albumin Yes11. Human albumin Yes 12. Porcine albumin US Pat. No. 4,299,726 Medium None 14. Sample 13 with lipoprotein cholesterol Yes15. 15. Cholesterol bound to surfactant None. 18. Phosphate buffered saline (PBS) None Lecithin None 19. With PBS with lipoprotein cholesterol

  From the above test results, it was determined that the addition of plasma substances to the washed erythrocytes allows lysis in the saponin lysis system. Albumin is thought to interact with erythrocytes and saponins to perform lytic effects. However, when washed red blood cells and bovine serum albumin are combined with other ingredients such as those disclosed in US Pat. No. 4,299,726, albumin does not cause lysis. However, dissolution occurs when lipoprotein cholesterol is added. Moreover, dissolution occurs when lipoprotein cholesterol is added to PBS.

  When using leukocyte analogs prepared from red blood cells as described above, it is preferred that the aqueous solution of plasma material be added to the hematological composition at least 12 hours prior to use in the device. When one or more leukocyte analogs are combined with lysable human erythrocytes to prepare a single multi-analytical reference plasma control product for a device that uses a lysis reagent, the aqueous solution of plasma material is bound cholesterol. Most preferably it comprises. A suitable example of the most preferred plasma substance is Moduquite ™ as described in US Pat. No. 4,290,774 (Miles, Inc.), which is a high density lipoprotein cholesterol combined with albumin. The final concentration of cholesterol in the suspending medium ranges from 400 to 1,200, preferably 600 to 1,000 mg / l, depending on the blood cell count in the final product.

  For control products using leukocyte analogs prepared by the preferred methods disclosed herein, red blood cells in the reference blood cell control product do not lyse efficiently when insufficient concentrations of cholesterol are used in the preferred medium. There is no noise and debris when using the saponin lysis reagent system because it does not lyse the cell membrane, and the leukocyte analog will have an average cell volume that is lower than the size required for the lysis reaction. If the medium contains a too high concentration of cholesterol, the red blood cells in the reference blood cell control product do not lyse efficiently and do not lyse the cell membrane, so there is no noise and debris.

  More particularly, using the control product in an instrument, such as an instrument using Coulter VCS type technology (which uses a reagent system as described in US Pat. No. 4,751,179), When distinguishing at least two populations of leukocytes, (1) lymphoid lineage (lymphocytes) and (2) myeloid cells (neutrophils, monocytes, eosinophils and basophils), the preferred suspending medium is Causes a reaction between the weaker lysis reagent and the non-fixed red blood cells, so that the red blood cells are lysed, but the leukocyte analogs are substantially unaffected, allowing the counting of each type of leukocyte analog. To do. As taught by US Pat. No. 4,751,179, the lysis reagent has two forms: (1) a lysis diluent containing saponin, which simultaneously dilutes a whole blood sample and removes its red blood cells Or (2) a two-part system composed of a lysis reagent containing a non-dissolved blood diluent followed by a saponin.

  When using prior art media such as those described in US Pat. No. 4,213,876, US Pat. No. 4,299,726, or US Pat. No. 4,358,395, the present specification The leukocyte analogs prepared by the preferred methods disclosed herein are less than the desired volume for the targeted leukocyte population. More particularly, when the preferred suspending medium is used with leukocyte analogs (prepared from red blood cells or leukocytes), the analog D.I. C. The volume is in the desired range for the targeted leukocyte population.

  In a more preferred embodiment, the suspending medium further comprises the addition of a nonionic surfactant. Surfactants have a high hydrophilic-lipophilic balance (HLB). The HLB typically has a value greater than 15, more preferably greater than 17. Typically, the surfactant is present in an amount effective to have a more specific lytic effect on red blood cells without adversely affecting the leukocyte analog. Furthermore, since the surfactant stabilizes free cholesterol in the control product, the cholesterol does not separate into the solution. As one skilled in the art will appreciate, an effective amount of surfactant can be determined empirically, but is typically less than 0.5% by weight of the control product.

Suitable nonionic surfactants include alkyl polyether alcohols of the general formula: R—X— (Y) n—H, wherein R is a lipophilic chain C ₈ -C ₁₈ carbon atom, where X is -O-,

-COO- a and and Y is _CH 2 _CH 2 O-or _{CH 2 CH 2 CH 2 O,} n is an integer of 15 to 50. Suitable commercial examples of these surfactants include Diazopan ™ SS-837 (GAF Chemical Corp.), Triton ™ X405 (Rohm and Haaas), and Pluronic FTM-127 PRILL (BASF Wyandotte Corp.).

  While not wishing to be bound by any theory, it is now believed that there is an interaction between red blood cells, weak lysing agents (eg, saponins), and plasma substances in the preferred suspension medium. More specifically, it is now believed that plasma substances affect cell membrane cholesterol, which further affects leukocyte analog responses to lytic reagents. Moreover, it is further believed that the surfactant makes the lysis reaction more specific for erythrocytes and does not adversely affect leukocyte analogs with respect to the measured control parameters. It is further believed that surfactants may also affect cholesterol found in cell membranes or plasma substances.

  Preferred suspending media for hematological reference control products with stability up to 6 months are plasma substances and any compatible fungicides and bactericides, and any adjuvants such as purine nucleosides, bile Solutes and cholic acid derivatives, phenothiazine compounds and their salts with antihistamine properties, and 4-aminobenzoic acid esters and derivatives and their salts with aesthetic properties, and spheronizing agents for erythrocytes, or combinations thereof An aqueous solution of Since one or more leukocyte analogs can be combined into a single reference blood cell control product for use with known lysing agents for erythrocytes, a preferred suspending medium formulation is leukocytes. It is the same for all analogs.

As those skilled in the art will appreciate, the suspending medium should have sufficient tonicity to avoid cell lysis. A preferred formulation for the suspending medium is as follows:
Suspension medium Approximate volume liter formulation 500ml distilled water
* 2. Propylparaben 0.3-1.0g
* 3. Methyl paraben 0.5-1.0g
* 4. Procaine hydrochloride 0.1-0.5g
* 5. Deoxycholic acid 0.1-0.9g
* 6. Lactose 10.0-50.0g
* 7. Actidione 0.1-0.6g
* 8. Trisodium citrate 2H ₂ O 3.0-8.0 g
* 9. Citric acid 1H ₂ O 0.3-0.9 g
* 10. Sodium dihydrogen phosphate 1H ₂ O 0.8-2.5 g
* 11. Fenergan hydrochloride 0.1-1.0g
* 12. Colistimate, sodium 0.2-0.9g
* 13. Penicillin G, sodium 0.5 × 10 ^{6 to} 3 × 10 ⁶ units * 14. Kanamycin sulfate 0.2-0.8g
* 15. Neomycin sulfate 0.2-1.0g
* 16.5'-AMP 0.4-1.0g
* 17. Adenine 0.2 ~ 0.8g
* 18. Inosine 0.4-1.0g
* 19. Dihydrostreptomycin sulfate 0.2-1.0 g
* 20. Tetracycline hydrochloride 0.2-1.0g
* 21.30% bovine albumin 100-350ml
22. Lipoprotein cholesterol 400-1,200 mg
23. Amount to make 1 liter of distilled water * Any optional component preferred to provide long-term stability to red blood cells and analogs.

The manufactured control product can be used to monitor and diagnose the cause of instrument dysfunction. In the present invention, a method has been developed that uses a hematology control product that can monitor and diagnose the instrument for problems related to:
1. Melting debris and noise 2. Set the reagent reagent pump volume. Laser alignment of instruments 4. 4. Setting instrument gain Flow rate mismatch in flow cell

This method provides a more specific indication of the type and cause of instrument dysfunction than non-specific flagging, which is diagnostic non-specificity or examination of test results by instrument operators provided by prior art methods. To do. In the following, a novel method using the previously described control product is described. As those skilled in the art will appreciate, the control product must contain lysable red blood cells to monitor cell debris and noise caused by invalid red blood cells. This method uses new control parameters of the physical properties of the control product to determine the cause of instrument dysfunction. These physical properties are selected from the group consisting of:
(1) D.E. C. Volume measured by current,
(2) high frequency (RF) magnitude,
(3) opacity, and (4) light scattering.

  The leukocyte population is used as an indicator of system operation. Preferably, a subpopulation of neutrophils and lymphocytes is used as a control parameter and measured to determine instrument performance. More preferably, a population of neutrophils is used as a control parameter and measured as an indication of system operation.

  The use of two control parameters is used to determine if there is a noise problem due to cell debris. Control parameters are counting ratio (COUNT RATIO) and elapsed time (ELAPSED TIME). The count ratio is a measure of the number of white blood cells in an analysis compared to the total count of events recorded in the analysis during a specified elapsed time. Elapsed time is usually measured in seconds. As the leukocyte count / total event count ratio approaches 100%, noise problems associated with cellular debris or instrument dysfunction are eliminated.

  One example using counting ratios in instruments using VCS technology is a comparison of the number of leukocyte analogs with the total count of 8192 particles obtained for a particular elapsed time. If this ratio is less than about 95%, there is an indication that noise or interference is a problem. At 95%, there is an indication that noise or jamming is a problem. The particular problem is further confirmed by additional tests that are further described in this disclosure.

  Another approach for monitoring and diagnosing the instrument for problems is to use continuous average fresh blood monitoring of the same set of statistically significant numbers of parameters of the patient's normal blood. When this average is outside the expected range of control parameters, there is an early indication of instrument dysfunction. Instrument dysfunction can be further assessed using the control products described herein.

One indication of whether the instrument is functioning properly is to determine the cell debris or noise measured by the instrument. Excess cell debris can be caused by incomplete lysis, temperature conditions or changes in reaction rate due to interference with the lysis reaction. Since various instrument systems have limitations on blood cell counts and data accumulation time, excess cell debris or noise from any source prevents proper acquisition and analysis of leukocyte populations. The cause of cellular debris or noise problems can contribute to several problems, including:
A. Conductivity noise due to sheath fluid and blood sample flow imbalance.
B. Lysis noise caused by inappropriate red blood cell lysis.
C. Flow noise caused by partial clogging, residue clogging or other problems.

  Measure each of the reagents added to the blood sample using a calibrated container to determine if the dysfunction is due to conductivity noise caused by a mismatch between the pump volumes of the sample flow To do. The volume of reagent transferred should be within a predetermined value range. If the reagent volume is not within the specified limits, adjust the reagent pump to increase or decrease the volume of the dispensed reagent.

  In an example of an instrument using VCS technology, the pump volume of the lysis reagent and quench reagent flow is measured. Each of these volumes is measured using a calibrated container. The volume of the reagent should be within a predetermined value range. If the reagent volume is not within the specified limits, adjust the reagent pump to increase or decrease the volume provided.

  A prepared control product containing an osmotically sensitive fixed blood cell analog population is further used to monitor the pump volume of the reagent. Changes in the volume of the lysis reagent will affect the final osmotic concentration of the diluted blood sample and will affect the measured volume of the analog population. Lower osmotic concentrations increase the measured volume of control blood cell analogs and higher osmotic concentrations decrease the measured volume of control blood cell analogs. Variations in the reagent pump volume that exceed the conductivity limits required to electrically balance the sheath fluid will generate noise in addition to changes in the volume of the control blood cells.

  Thus, after conductivity noise has been minimized by the method described above, lysis noise resulting from inadequate erythrocyte lysis will result in a known number of stabilized red blood cells and at least one leukocyte analog cell population. Inspected using the containing control product. If lysis is a problem, the control blood cells will show a count ratio lower than about 95% of the maximum count ratio. Furthermore, although the volume of the reagent can be adjusted by the methods described above, the kinetics of dissolution with respect to the temperature of the dissolution reaction will be affected. More particularly, when the room temperature of a laboratory instrument is 50 ° F., the lysis reaction requires a different lysis reagent and quench reagent than when the temperature is 90 ° F. to minimize lysis noise. It was discovered.

  In an example of an instrument using VCS type technology, a lysis reagent and a quench reagent are reacted with a blood sample to form a lysed and diluted suspension of leukocytes suitable for measurement. In a VCS type device, blood cells are reacted with a lysis reagent to swell hypotonic blood cells, lyse red blood cells and platelets, and lyse red blood cells and platelet membranes. Reaction with the quench reagent stops swelling and lysis and initiates the process of re-equilibration of leukocytes to the natural size of leukocytes. Due to the nature of the reaction, the lysis and quench reagents are very different in both osmotic concentration and conductivity. The final osmotic concentration and conductivity of the sample stream is proportional to the volume, osmotic concentration and conductivity of the lysis reagent, quench reagent and blood sample.

  Two control parameters are used to determine if the reagent pump volume is within specification or to determine the cause of the dysfunction. Monitoring the count ratio and neutrophil DC MEAN facilitates the differentiation of changes in pump volume within and beyond the conductivity tolerance. The DC average of neutrophils is the average value of neutrophils in the DC channel.

  An approach to monitoring and diagnosing the instrument for problems is to use the same set of consecutive average fresh blood monitoring of a statistically significant number of parameters of the patient's normal blood. When this average is outside the expected range of control parameters, there is an early indication of instrument dysfunction. Instrument dysfunction can be further assessed using the control products described herein.

  It has been discovered that the white blood cell count ratio is related to the reagent pump volume calculated from the concentration of the known reagent and the measured pump volume, expressed as the conductivity or osmotic concentration of the dilution. FIG. 1 represents a comparison of white blood cell count ratios compared to the conductivity of the diluted blood sample in the flow of diluted blood sample to be tested. As shown in FIG. 1, as the quench reagent volume increases, the counting ratio increases plateau. As the quench volume increases beyond the optimal range, the counting ratio decreases substantially.

  To obtain comparable Figure 1 using the control product, prepare a volume of lysis reagent in the first volume expected to provide adequate lysis. The quench volume is adjusted from preparing insufficient quench reagent to preparing excess amount of quench reagent. When the quench reagent volume is insufficient, the counting ratio is significantly reduced. As the quench volume increases, the counting ratio increases and a plateau above the optimum operating range increases the quench volume above the optimum range, substantially reducing the counting ratio. Once this information is obtained, the quench reagent pump can be adjusted to obtain the quench reagent volume in the middle of the plateau.

  After the quench volume is adjusted, the lysis reagent pump can be readjusted. It was discovered that the DC average of the control product neutrophils is related to the pump volume of the lysis reagent. Monocyte populations show similar sensitivity to neutrophil populations. The lysis reagent pump is adjusted to obtain an osmotic concentration corresponding to the DC average of neutrophils measured before the control product. Previously measured neutrophil DC averages are obtained as assay values from a reference instrument.

  FIG. 2 shows a comparison of the DC average of neutrophils after dilution of the blood sample with lysis and quench reagents and compared to the osmotic concentration of the blood sample to be analyzed. As shown in FIG. 2, as the osmotic concentration increases, the neutrophil DC average of the control product decreases.

  Two control parameters, white time (WHITE TIME) and date and time (DATE TIME), are used to determine if there is a problem due to remaining or partial clogging. These two control parameters facilitate the determination of these types of problems. White time is the product of the number of leukocyte analogs and the time required for analysis. The date and time is the date when the control sample was analyzed. It has been found that using these two control parameters, along with a control product containing a known number of leukocyte analogs, provides information on instrument operation issues.

  In the method of the invention, the control product is used at least daily. Record white time and date and time to get meaningful information about the existence of flow problems. More specifically, if the white time is graphed with respect to date and time, a two-dimensional graph of instrument motion can be obtained to determine the trend of instrument flow behavior.

  In VCS-type instruments, another method is available for examining the sample flow rate. The VCS instrument records the white time and date and time for each patient blood sample analyzed therewith. By comparing these control parameters for a statistically significant number of patient blood samples, an extended database can be obtained to monitor the history of instrument flow behavior. More particularly, by recording the white time and date and time for each patient's blood sample, it can be determined whether any remaining clogging or partial clogging has occurred. Residual clogging may limit subsequent sample flow, but may not produce a detectable characteristic of blood cell count / second. Partial blockage may increase the time required to obtain a white blood cell count because it reduces the flow of blood cells through the flow cell.

  In addition, when monitoring these parameters on patient samples, to find out if the flow rate has been adversely affected and whether the level of noise and debris interferes with the expected time to obtain a white blood cell count A two-dimensional analysis can be obtained. Excess debris will reduce the amount of time required to obtain a specified number of white blood cells. This two-dimensional analysis provided an improvement over the complex multi-dimensional analysis of the prior art.

  The prior art method of determining problems with flow cell accuracy has been to measure the number of white blood cells detected during a predetermined time. More particularly, the instrument operator uses a specially selected fresh blood with a known number of white blood cells and measures the time it takes for the instrument to detect white blood cells. It was estimated that there was no flow cell problem if the instrument detected white blood cells within the specified time. However, this measurement only provided information about the instrument for a particular analysis. This measurement did not provide meaningful information about instrument operation over an extended period of time. More specifically, the white blood cell count / unit time graph only provides a momentary observation of instrument operation for the day. In order to obtain observations of instrument movement over several days, a three-dimensional change in the results was necessary. Moreover, the method of the present invention uses a control product with a known number of blood cells, thereby eliminating the need to obtain specially selected fresh blood and to determine the number of white blood cells it contains.

  In addition, the control product can be used to monitor the instrument to determine if there is a problem using the instrument's laser, laser optics or laser alignment. The laser alignment has three dimensions corresponding to X, Y and Z.

  Currently, the material used to set and then monitor laser alignment and gain adjustment is latex microparticles. A method that uses latex particles is to monitor the mean of the population and the coefficient of variation of the latex population. However, if the laser lens accumulates dust or other debris that obscures the signal of the laser beam, prior art implementations will increase the gain of the laser to overcome interference with the laser beam. Allowed clarity. Furthermore, latex particles are not responsive to the reagents used in the instrument. Moreover, latex particles are another control product that is required to work in a calibrated mode in the instrument. Latex particles require additional work to be performed to monitor instrument operation. Accordingly, it would be advantageous to have a single hematological control product that responds to reagents used in the instrument and provides an indication of laser operation in the instrument.

  The X-axis alignment is the most sensitive axis and is determined to produce the greatest effect on the location and distribution of light scatter in the control product. When the X axis is properly aligned and the distribution width is minimal, the rotated light scattering position of the control product is at its maximum. As the alignment decreases to a slight misalignment and then to a large misalignment, the position of the control product in the histogram shifts to the left, the average value decreases, and the channel distribution of the rotated light scatter broadens.

  When fresh blood is analyzed and X-axis misalignment is present, the leukocyte population is shifted to the left (low light scatter), resulting in loss of counted leukocytes and increased noise. When the misalignment becomes more extreme, the effect on the analysis of fresh blood is that the white blood cell subpopulation is immersive and the discrimination is lost.

  Y-axis and Z-axis misalignment does not show a change in the position of the same type of white blood cell population exhibited by white blood cells when caused by X-axis misalignment. It has been determined that the Z-axis alignment is relevant in determining whether the lens is in the proper focus where the lens provides the sharpest image. If the Z axis is not in the correct alignment, the resulting scatter plot will not show a clear and distinct cluster of leukocytes. More specifically, in the light scatter / DC volume histogram, the separation of lymphocyte and monocyte populations from neutrophil populations may not be clear. There will be a decrease in the separation of the light scattering peaks of the volume population. More specifically, there will be a decrease in amplitude in individual populations and an increase in valley amplitude between populations.

  It was found that the control product lymphocyte population provides information to determine low light scatter, while the neutrophil population provides information to determine high light scatter. The relationship between the location of these two populations is a system that produces both a bias and a proportional effect on the population location, eg, laser alignment (bias) and adjustment of DC, RF and LS gain (proportional). Monitor for fluctuations.

  The use of two control parameters can be used to determine problems due to laser operation. Monitoring of the neutrophil RLS average (NEUTROPHIL RLS MEAN) and the ratio of PEAK TO VALLEY RATIO will provide an indication of laser operation and X-axis and Z-axis alignment. The lymphocyte RLS average (LYMPHOCYTE RLS MEAN) shows a sensitivity similar to the neutrophil RLS average. The neutrophil RLS average is the average value of neutrophils in the light scattering channel. The lymphocyte RLS average is the average value of lymphocytes in the light scattering channel. The peak-to-valley ratio (PVR) is the amplitude of the rotated light scatter signal of the neutrophil population / valley rotated light scatter signal formed between the lymphocyte and monocyte population and the neutrophil population. Ratio.

  PVR provides reliable quality control parameters for use in a method for determining whether a laser problem exists. When using control products, the ratio of mountain to valley should satisfy a predetermined limit. If the PVR meets or exceeds this number, the laser is operating optimally. More specifically, a PVR that satisfies a predetermined number indicates that there is a clear separation of the neutrophil population and the leukocyte and monocyte population. If the PVR is less than a predetermined number, there is an indication that there is a laser problem.

  As previously mentioned, prior art implementations allowed an increase in laser gain to compensate for dirty lenses. However, latex particles do not provide any specific information regarding misadjustment of the gain of the light scattering signal when optical disturbances are present. When the gain adjustment was inappropriately increased, it was determined that the position of the entire neutrophil population in the rotated light scattering histogram encompassing the neutrophil RLS average shifts to the right.

  In accordance with the method of the present invention, if it is determined that the PVR is less than the predetermined value and the neutrophil RLS average is greater than the predetermined value, the instrument is optically disturbed or X-axis or Z-axis aligned. Is shown to have problems. Thus, the technician will know to clean the lens and calibrate the laser alignment. In addition, if it is determined that the determined PVR is smaller than the predetermined value and the neutrophil RLS average is smaller than the predetermined value, the instrument has an optical disturbance that was not compensated for by the gain increase. Or it is shown to have a problem due to X-axis or Z-axis alignment. Moreover, if the PVR is within the predetermined value range and the neutrophil RLS average is greater than or less than the predetermined value, it indicates that the laser gain is set improperly. Still further, if the PVR is less than the predetermined value and the neutrophil RLS average is within the predetermined value range, the laser has lost power and requires a laser replacement.

The above specification describes the use of some control parameters to determine whether the instrument is operating according to the manufacturer's specifications, and the use of control parameters to diagnose the cause of dysfunction However, Table 1 provides a detailed analysis and its control parameters can be used for statistically significant values of control products or patient blood samples to detect and diagnose problems with the instrument system. it can. In this table, the following abbreviations are used:
s = sensitivity for applicable instrument function SD = standard deviation OP = opacity x = measurement of control product or sample

  In addition, polystyrene or other plastic beads can be added to the control product to prepare a single reference control product that can further demonstrate instrument operation. More specifically, using this type of control product along with control parameters can provide information about the operation of the instrument. For example, if the latex particle DC gain is within specification, but the control product has a DC average outside the specification range, there is an indication that there is a problem with the reagent pump volume.

  When this type of control product is used in an instrument using VCS technology, the above example shows that there is a problem with the volume of lysis reagent or quench reagent. More specifically, in a VCS-type instrument, if the latex particle DC gain is within specification, but the control product has a DC average above the manufacturer's specification, then the volume of lysis reagent is greater than the optimal level, There is an indication that the volume of the quench reagent is less than the optimum level. Still further, in the VCS example, if the latex particle DC gain is within specification but the control product has a DC average below the manufacturer's specification, the volume of lysis reagent is less than the optimal level or quench reagent There is an indication that the volume of is greater than the optimum level.

  Methods for preparing leukocyte analogs for use in the methods of the invention are described in the examples below. It should be understood that Example 1 is a specific example of preferred reagents and techniques for treating cancer cells, and that the formulation is merely illustrative of what can be used in the methods of the invention. It should be understood that Examples 2, 3, and 4 are specific examples of preferred reagents and techniques for treating alligator cells, and that the formulations are merely illustrative of what can be used in the methods of the invention. . Example 5 shows an enzyme that uses human leukocytes to generate five subpopulations of leukocyte analogs, and it should be understood that the formulation is merely illustrative of what can be used in the methods of the invention. Example 6 shows the assembly of four leukocyte populations. It should be understood that these examples provide formulations for leukocyte analogs that are merely exemplary.

The reagents and / or techniques described are also applicable to blood cells from animals other than cancers and alligators. Other ingredients and ratios can be used in accordance with this disclosure.
Example 1
Lymphocyte analogs from cancer erythrocytes The following are specific examples of preferred reagents and recommended specific procedure steps for treating cancer erythrocytes to obtain animal-selected lymphocyte analogs. is there. It should be understood that the formulations and techniques are merely exemplary, and that other components, ratios and techniques can be used in accordance with this disclosure in the present invention.
Phosphate buffered saline (PBS)
Liter formulation 1.1 Hydrochloric acid sodium phosphate: 0.2g
2.2 Hydrochloric acid sodium phosphate 7H ₂ O: 2.0 g
3. Sodium azide: 0.1g
4). Sodium chloride: 9.4g
5). Make up to 1 liter with distilled water: pH approximately 7.4; osmotic concentration 315-345 mOsm / kg.
Leukocyte hypotonic solution Basic sodium phosphate: 0.2g
2.2 Basic sodium phosphate 7H ₂ O: 2.0 g
3. Make up to 1 liter with distilled water: pH approximately 7.8; osmolarity 15-25 mOsm / kg.
Method 1. Avian red blood cells having an average cell volume range of about 140-170 fL are selected. The filled avian erythrocytes are washed with phosphate buffered saline (PBS).
2. Add 1.0-5.0 mg of cholesterol to a 2 × 10 ⁶ / μ1 blood cell count and incubate at room temperature for 2-6 hours.
3. A glutaraldehyde fixing reagent having a glutaraldehyde content of about 0.1-0.8% is prepared by adding a commercial 25% glutaraldehyde product to a chilled leukocyte hypotonic solution. Preferably, the temperature is 2-8 ° C. A preferred concentration of glutaraldehyde is approximately 0.35%.
4). Washed red blood cells are added to the measured amount of Step 3 fixative at a 1:35 dilution. Transfer to a sealed container and slowly rotate the container at 2-8 ° C. for 18-24 hours. The decrease in hemoglobin content is calculated to be approximately 60% by weight.
5). The supernatant fluid is removed and the blood cells are washed several times with PBS and resuspended in a suitable storage solution.
6). In order to leave the lymphocyte analog alone, the washed and fixed blood cells are resuspended in the suspension medium of the present invention and the concentration is adjusted to simulate the concentration of human lymphocytes in normal human blood .
7). Blood cells washed and fixed in the suspending medium of the present invention having the desired analogs for other hematological compositions and multi-parameter hematology control products for multi-hematologic parameters for control products And a blood cell count is appropriate for determining the ratio of lymphocytes.
8). Using appropriate stabilizers, fixed blood cells can be stored for a period of more than 6 months.

In accordance with the above example, comparable results are obtained using other types of mammalian red blood cells.
Example 2
Monocyte Analogs from Alligator Red Blood Cells The following are specific examples of preferred reagents and recommended specific procedure steps for treating alligator red blood cells to obtain monocyte analogs. It should be understood that the formulations and techniques are merely exemplary, and that other components, ratios and techniques can be used in accordance with this disclosure in the present invention.
Monocyte hypotonic solution 1.1 Basic sodium phosphate: 0.1 g
2.2 Basic sodium phosphate: 1.0 g
3. Make up to 1 liter with distilled water: pH approximately 7.8; osmotic concentration 5-15 mOsm / kg. Wash solution for blood cells (PBS), as described in Example 1.
Method 1. Alligator red blood cells having an average cell volume range of about 350-450 fL are selected. Wash the filled alligator red blood cells with PBS.
2. Add 1.0-5.0 mg cholesterol to 1 × 10 ⁶ / μ 1 blood cell count and incubate at room temperature for 3-5 hours.
3. A glutaraldehyde fixing reagent having a glutaraldehyde content of about 0.1-0.8% is prepared by adding commercial 25% glutaraldehyde product to a cooled monocyte hypotonic solution. Preferably, the temperature is 2-8 ° C. A preferred concentration of glutaraldehyde is approximately 0.15%.
4). Washed red blood cells are added to the measured amount of Step 3 fixative at a 1:50 dilution. Transfer to a sealed container and slowly rotate the container at room temperature for 18-24 hours. The decrease in hemoglobin content is calculated to be approximately 40% by weight.
5). The supernatant fluid is removed and the blood cells are washed several times with PBS and resuspended in a suitable storage solution.
6). In order to leave the monocyte analog alone, washed blood cells are resuspended in the suspension medium of the present invention and the concentration is adjusted to simulate the concentration of human monocytes in normal human blood. To do.
7). Blood cells washed and fixed in the suspending medium of the present invention having the desired analogs for other hematological compositions and multi-parameter hematology control products for multi-hematologic parameters for control products At a concentration suitable for monocyte measurement.
8). Using appropriate stabilizers, fixed blood cells can be stored for a period of more than 6 months.

Example 3
Eosinophil analogs from alligator erythrocytes The following are specific examples of preferred reagents and recommended specific procedure steps for treating alligator erythrocytes to obtain eosinophil analogs. It should be understood that the formulations and techniques are merely exemplary, and that other components, ratios and techniques can be used in accordance with this disclosure in the present invention.
Monocyte hypotonic solution 1.1 Basic sodium phosphate: 0.32 g
2.2 Basic sodium phosphate: 8.08 g
3. Make up to 1 liter with distilled water: pH approximately 8.0; osmotic pressure 75-85 mOsm / kg.
Eosinophil hemoglobin denaturation treatment solution Dimethyldicocoammonium chloride: 2.5g
2. Tris (hydroxymethyl) aminomethane: 6.06 g (organic buffer)
3. Bring to 1 liter with distilled water: pH approximately 10.5.
Eosinophil post-treatment cleaning solution Polyoxyethylated alkylphenol: 5 g (Dazopan ™ SS-837, GAF Chemical Corporation)
2. Make up to 1 liter with distilled water.

Wash solution for blood (PBS), as described in Example 1.
Method 1. Alligator red blood cells having an average cell volume range of about 400-500 fL are selected. Wash the filled alligator red blood cells with PBS.
2. Add 0.25-1.25 mg cholesterol to a 1 × 10 ⁶ / μ1 blood cell count and incubate at room temperature for 2-5 hours.
3. A glutaraldehyde fixing reagent having a glutaraldehyde content of about 0.1-0.8% is prepared by adding a commercial 25% glutaraldehyde product to an eosinophil hypotonic solution. The preferred concentration of glutaraldehyde is approximately 0.2%.
4). Washed red blood cells are added to the measured amount of Step 3 fixative at a 1:50 dilution. Transfer to a sealed container and slowly rotate the container at room temperature for 18-24 hours.
5). The supernatant fluid is removed and the blood cells are washed several times with PBS.
6). Washed red blood cells are added to the eosinophil hemoglobin denaturing treatment solution at a 1:10 dilution. Transfer to a sealed container and slowly rotate the container at room temperature for 2-4 hours.
7). The supernatant fluid is removed and the blood cells are washed several times with an eosinophil post-treatment washing solution to remove the eosinophil hemoglobin denaturing treatment solution. It is then suspended in a suitable storage solution.
8). In order to leave the eosinophil analog alone, the washed and fixed blood cells are resuspended in the suspension medium of the present invention and the concentration adjusted to the concentration of human eosinophils in normal human blood. Simulate.
9. For multi-hematologic control products, blood cells washed and fixed in the suspending medium of the present invention having the desired analog to other hematological compositions and multi-parameter hematology control products are treated with eosinophils. Add at a concentration suitable for sphere measurements.
10. Using appropriate stabilizers, fixed blood cells can be stored for a period of more than 6 months.

Example 4
Neutrophil analogs from alligator erythrocytes The following are specific examples of preferred reagents and recommended specific procedure steps for treating alligator erythrocytes to obtain neutrophil analogs. It should be understood that the formulations and techniques are merely exemplary, and that other components, ratios and techniques can be used in accordance with this disclosure in the present invention.
Neutrophil hypotonic solution 1.1 Basic sodium phosphate: 0.23 g
2.2 Basic sodium phosphate: 5.32 g
3. Make up to 1 liter with distilled water: pH approximately 8.0; osmotic pressure concentration 45-65 mOsm / kg.

Wash solution for blood cells (PBS), as described in Example 1.
Method 1. Alligator red blood cells having an average cell volume range of about 400-500 fL are selected. Wash the filled alligator red blood cells with PBS.
2. A glutaraldehyde fixing reagent having a glutaraldehyde content of about 0.1-0.8% is prepared by adding a commercial 25% glutaraldehyde product to a neutrophil hypotonic solution. The preferred concentration of glutaraldehyde is approximately 0.4%.
3. Washed red blood cells are added to the measured amount of Step 3 fixative at a 1:50 dilution. Transfer to a sealed container and slowly rotate the container at room temperature for 18-24 hours.
4). The supernatant fluid is removed and the blood cells are washed several times with PBS and resuspended in a suitable storage solution.
5). The filled blood cells are added to the nonionic surfactant solution. The solution tends to normalize the volume of donor blood cells. This solution comprises 0.5 g of octylphenoxy polyethoxyethanol (Triton ™ X-100, Rohm and Haas Co.) with 0.5 g of approximately 13.5 HLB in 1 liter of distilled water.
6). The supernatant fluid is removed and the blood cells are washed several times with PBS and resuspended in a suitable storage solution.
7). In order to leave the neutrophil analog alone, the washed and fixed blood cells are resuspended in the suspension medium of the present invention and the concentration adjusted to the concentration of human neutrophils in normal human blood. Simulate.
7). For multihematologic control products, neutrophils washed and fixed in the suspension medium of the present invention having the desired analog to other hematology compositions and multiparameter hematology control products are Add at a concentration suitable for sphere measurements.
8). Using appropriate stabilizers, fixed blood cells can be stored for periods exceeding 6 months.

Example 5
Five subpopulations of leukocyte analogs from human leukocytes Whole blood is added to a dextran (molecular weight 100,000-50,000) solution at a 1:20 dilution and allowed to settle for 1-3 hours by gravity.
2. Remove the supernatant containing white blood cells, platelets and some residual red blood cells.
3. Centrifuge the product of step 2 at about 300 RCF for about 10 minutes. Platelets are aspirated, leaving a button of white blood cells, residual red blood cells, and a small amount of plasma to resuspend the blood cells.
4). A suitable lysing agent such as water is added to lyse the red blood cells from the white blood cells.
5). Centrifuge the product of step 4 and remove the supernatant, leaving the filled white blood cells. The filled leukocytes are resuspended in approximately equal volumes of saline.
6). White blood cells are added at a 1:10 dilution in a suitable isosmotic fixation solution, such as 5% formaldehyde and 95% PBS (volume%), transferred to a sealed container, and the container is slowly rotated at room temperature for 18-30 hours. To do.
7). A glutaraldehyde human fixing solution with approximately 0.1% concentration of glutaraldehyde is added to the pool at a 1: 1 dilution and the fixation is continued for another 8-12 hours.
8). The supernatant fluid is removed and the blood cells are washed several times with PBS and resuspended in a suitable storage solution.
9. To leave the assembly of the five sub-populations of leukocyte analogs alone, the washed and fixed blood cells are resuspended in the suspending medium of the present invention and the concentration adjusted to adjust in normal human blood. Simulate the concentration of human leukocytes.
10. For multi-hematologic control products, add blood cells washed and fixed in the suspending medium of the present invention having the desired analogs to other hematological compositions and multi-parameter hematology control products; A blood cell count is suitable for measuring the white blood cell ratio.
11. Using appropriate stabilizers, fixed blood cells can be stored for a period of more than 6 months.

Example 6
The following amounts of individual components are used in a subassembly to simulate the targeted composition of leukocytes in normal human blood samples:
Stock solution 0.150 L Example 1 Lymphocytes 500 × 10 ³ / μl
0.040 L Example 2 Monocyte 500 × 10 ³ / μl
0.030 L Example 3 Eosinophils 500 × 10 ³ / μl
0.280 L Example 4 Neutrophils 500 × 10 ³ / μl
0.500 L Diluent Phosphate buffered saline In the final assembly of the four leukocyte populations, the supernatant fluid is removed and the blood cells are then re-introduced into 1.0 liter of an aqueous solution of Moduite ™ with a final concentration of 800 mg cholesterol. Suspend.

  The assembly can be stored for up to about 6 months with the addition of known suitable stabilizers.

  The ratio and total blood count for the leukocyte population can be adjusted to represent pathological conditions in human blood as well as normal conditions. These compositions are equally effective in control and calibrating products, especially for automated particle analyzers that use the Coulter principle. In addition, latex particles can be added to the control product to form a single control product that can further direct instrument operation.

  Thereafter, counts of untreated human red blood cells, simulated white blood cells, and stabilized or simulated platelets at a ratio such that the final red blood cell, white blood cell and platelet counts and hemoglobin content and hematocrit fall within the desired range. A suspension can be added.

Stabilized platelets are prepared by methods known in the art. Effective methods include the following:
1. Combinations of iodoacetamide and iminodiacetic acid or their solutes and compatible bacteriostatic agents in aqueous solutions maintained at a preselected range of pH and osmotic concentrations, as described in US Pat. No. 4,405,719. ing.

  2. Fixative-stabilized composition containing a nonionic surfactant that is a mixture of glutaraldehyde at a concentration of 0.1 to 5% and an ethoxylate of certain isomeric linear alcohols, US Pat. No. 4, 389,490, for further details.

  3. Human platelet analog comprising red blood cells optionally stabilized, bound and formulated to have a size range and volume stability close to that of human platelets, as described in US Pat. No. 4,264,470 Has been.

Values can be varied for each of the hematological parameters to represent abnormally low and abnormally high conditions. The leukocyte count in normal blood is 5,000-11,000 / microliter (μl), the lymphocyte value is 20-40%, the mononuclear cell value is less than 10%, the granulocyte value Is 60-80%, the value of eosinophils is approximately 5%, and basophils are less than approximately 2%. The normal range in human blood for erythrocytes is 4,000,000 to 5,000,000 blood cells / μl. The value of normal hemoglobin is 12-16 g / 100 ml. The term “hematocrit” is defined as the ratio of packed red blood cell volume / white blood cell volume. The normal ratio in humans is about 45%. Mean blood cell value is the ratio of filled red blood cell volume (ml / liter blood) / red blood cells (10 ⁶ / μl). The average blood hemoglobin concentration is an index (%) indicating the average weight of hemoglobin per 100 ml of packed red blood cells. Mean blood hemoglobin is the ratio of hemoglobin content (g / l) / red blood cells (10 ⁶ / μl).

  Although the invention has been described in detail in the foregoing specification for purposes of illustration, various changes and modifications may be made in the details herein without departing from the spirit and scope of the invention.

Comparison of the white blood cell count ratio compared to the conductivity of the diluted blood sample in the core to be tested. Comparison of neutrophil DC average compared to osmotic concentration of blood sample to be analyzed after dilution with lysis and quench reagents.

Claims (7)

a. At least one leukocyte subgroup analog, wherein said leukocyte subgroup analog is derived from blood cells that have been treated to be resistant to degradation by a lysis reagent used in hematological testing procedures; And the analog remains responsive to the reagents used in the operation of the instrument, and b. Plastic beads,
A hematology control product for determining a dysfunction of a hematology instrument that analyzes blood based on DC volume and / or light scatter measurements , wherein the dysfunction is a light scatter measurement At least one dysfunction selected from the group consisting of accumulation of dirt or other debris that obscures the signal of the laser beam of the lens of the instrument to affect and variation of the pump volume of the reagent that affects DC volume measurement A hematological control product .   The hematological control product of claim 1, wherein the leukocyte subpopulation analog comprises at least one neutrophil analog.   The hematological control product of claim 1, further comprising the addition of lysable red blood cells.   The hematological control product of claim 1, wherein the control product further comprises at least three different leukocyte subgroup analogs.   The hematological control product of claim 4, wherein the control product further comprises an aqueous solution of a plasma substance.   6. A hematological control product according to any one of claims 1 to 5, wherein the analog is derived from red blood cells.   The hematological control product according to any one of claims 1 to 6, wherein the beads are made of latex, polystyrene or other plastic material.

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