WO1985005684A1 - Method and reagent system for four-population differential determination of leukocytes - Google Patents

Method and reagent system for four-population differential determination of leukocytes

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Publication number
WO1985005684A1
WO1985005684A1 PCT/US1985/000868 US8500868W WO8505684A1 WO 1985005684 A1 WO1985005684 A1 WO 1985005684A1 US 8500868 W US8500868 W US 8500868W WO 8505684 A1 WO8505684 A1 WO 8505684A1
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WO
WIPO (PCT)
Prior art keywords
reagent
saponin
volume
reagent system
lytic
Prior art date
Application number
PCT/US1985/000868
Other languages
French (fr)
Inventor
Stephen L. Ledis
Harold R. Crews
Ted Sena
Original Assignee
Coulter Electronics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Coulter Electronics, Inc. filed Critical Coulter Electronics, Inc.
Priority to DE8585902786T priority Critical patent/DE3587036T2/en
Publication of WO1985005684A1 publication Critical patent/WO1985005684A1/en
Priority to KR1019860700056A priority patent/KR860700153A/en

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Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5094Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/10Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/101666Particle count or volume standard or control [e.g., platelet count standards, etc.]
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/10Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/107497Preparation composition [e.g., lysing or precipitation, etc.]

Definitions

  • saponin The natural product known as saponin has long been used as a red blood cell lytic agent.
  • Saponin is chemically defined as a class of glycosides of various mono- or polysaccharides, with steroid or triterpene alcohols.
  • Quillaja saponin is isolated as a natural ' product from quillaja tree bark. This saponin has detergent-like properties and hemolyzes red blood cells when used at very low concentrations compared to synthetic ionic and nonionic hemolytic agents.
  • the chemical treatment, structure and purity of commercial quillaja saponin is not generally specified or tested, and the material does vary from lot to lot.
  • the invention was materially expanded in U. S. Patent 3,502,974, Coulter et al, 1970, using radio frequency (RF) current in addition to DC current field excitation to provide not only DC volume information concerning the particle studied, but also information due to the composition and nature of the material constituting the particle.
  • RF radio frequency
  • This patent discloses apparatus capable of distinguishing between particles of identical size, but of different material. By generating the particle sensing field by means of both a low frequency or direct current (DC) and radio frequency (RF) current excitation, two or more interrelated output signals can be derived from the passage of a single particle through the electrical field.
  • the subject particles are nearly always insulators with respect to low frequency or direct current fields, they are capable of carrying or impeding radio frequency current differently from the surrounding electrolyte. This may be due to differences in the dielectric constant in the case of homogeneous particles, or to the sac-like structure in the case of blood cells which have, enclosed in an extremely thin membrane, contents having conductivities different from the electrolyte. Thus, while all the DC current goes around a blood cell, some of the RF current will go through it.
  • opacity is defined as the RF impedence divided by the DC impedence.
  • the relative electrical opacity of a particle becomes an identifying feature of the particle- contents, and hence, its particle type for classification purposes. To the extent that different types of particles each possess a different opacity, the difference between them is detectable. However, significantly different particles ' can possess substantially the same opacity and such particles cannot be classified effectively in this manner. In U. S. Patent 3,836,849, 1974, Coulter et al taught that it is possible to change selectively the opacity of particle types by treatment of the particles, so that detectable differences result.
  • red blood cells and white blood cells nominally have different sizes, their size ranges tend to overlap, or at least under certain conditions of health could overlap. Moreover the opacities of these two types of blood cells may also overlap.
  • a cytological fixing agent which is a monoaldehyde such as formaldehyde, is added to a blood sample.
  • a hemolyzing agent then is added after the fixation step to cause the red blood cells to release their hemoglobin content into solution.
  • Addition of a specific cytochemical substrate, chromogenic precipitating coupling reagent, and pH buffer causes deposition of an insoluble dye in a specific type of cell containing an immobilized enzyme.
  • the solution containing the dyed blood cells then is passed through a photometric counter.
  • cytological fixing solution utilized only a monoaldehyde. Dialdehydes are stated to be unsuitable, since they cross-link and produce extracellular precipitates.
  • a preferred way to solve the problem is to hemolyze the red blood cells by addition of a reagent to the suspension of cells to cause the red blood cells to rupture and release their hemoglobin content into the solution.
  • Ledis et al U. S. Patent 4,286,963, 1981, teaches a method for two-volume analysis of leukocytes using a COULTER COUNTER analyzer which employs only DC field excitation instrumentation and quaternary ammonium salts as lysing agents.
  • Ledis et al, U. S. Patent 4,485,175, to Coulter Electronics, Inc. concerns a method and reagent system for three-volume differential determination of lymphocyte, monocyte, and granulocyte populations of leukocytes, using quaternary ammonium salts as lysing agents and the COULTER COUNTER Model S Plus automated blood counter, which instrument employs only direct current field excitation.
  • Other dye compositions for differential analysis of white blood cells include a hypotonic aqueous solution of a metachromatic fluorochrome dye such as acridine orange, Adams, U. S. Patent 3,883,247.
  • the white cell analysis is made by suspending a sample of fresh blood in the dye solution, subjecting the suspension, before dye uptake equilibrium is reached, to radiation from a light source, e.g. radiation from a blue laser, having a wave length within the range of absorption of the dye, and distinguishing the white cells from other blood particles by detecting fluorescences, e.g. green vs. red fluorescences.
  • Fluorescent dyes suitable for specifically dyeing eosinophil granules are the anilino or toluidino naphthalene sulfonic acids and their alkyl, alkoxy or halogen substituted derivatives.
  • instrumentation and fluorochromes for automated multiparameter analysis of cells is further described by R.C. Leif et al. in Clinical Chemistry, Vol. 23, pp 1492-98 (1977).
  • Eosinophils have been observed also by enzyme staining such as by the Technicon Hemalog D and H6000 instruments, (Ansley and Ornstein, Adv. Automated Anal., Vol. 1 437 (1971), Kaplow, Macrophages and Lymphocytes, Part A, 211 Plenum (1980).
  • This invention concerns a reagent system and a method for classifying and counting at least four populations of leukocytes using flow analysis instrumentation. More particularly, the present invention relates to a multicomponent reagent system for rapidly eliminating the red blood cells in a sample of whole blood and for maintaining and modifying leukocytes in a manner suitable for flow analysis classification into four major categories which have been identified as (1) lymphocytes, (2) monocytes, (3) neutrophils and (4) eosinophils.
  • the reagent system comprises aqueous solutions of: (A) a lytic diluent containing saponin; or a blood diluent which is followed by a lytic reagent containing saponin; (B) a fixing reagent containing a cross-linking compound such as glutar ldehyde, each of these two components being maintained at a predetermined concentration, pH and osmolality.
  • This method is rapid, reliable and effective for normal and abnormal bloods and blood control material.
  • Preservatives can be added to inhibit the growth of microorganisms.
  • Other additives also can be included.
  • a fluorescent dye is included in the lytic diluent when studies make a determination by fluorescence.
  • the lysed and fixed blood sample is brought to the proper concentration for flow analysis using a diluent which also can contain potassium ferricyanide and potassium cyanide for converting the hemoglobin to a chromagen suitable for hemoglobinometry.
  • This invention relates also to a method for discriminating of major categories of leukocytes by flow instrumentation which measures two or more of the primary parameters of DC volume, RF size, fluorescence and light scatter, and also provides histograms based on selected mathematical combinations of the primary parameters.
  • FIGURES 1 through 5 illustrate the leukocyte subpopulation identification accomplished by the method of this invention.
  • the encircled areas represent cell populations or cell clusters that are generated by accumulation of data points, in which each data point is determined by coordinates which are proportional to certain cell parameters. This form of diagram is known as a cytogram.
  • the hereinafter presented Examples explain each FIGURE.
  • the purpose of this invention is to provide a method for the rapid lysing of the red blood cells in whole blood in a manner that preserves and/or modifies the leukocytes, so that they can be distinguished or classified into subpopulations.
  • whole blood is treated with a lysing reagent.
  • This lysing reagent has two forms: (D a lytic diluent containing saponin, which simultaneously functions to dilute the whole blood sample and stromatolyse its red blood cells; or (2) a two part system comprised of non-lytic blood diluent followed by a lytic reagent containing saponin.
  • the lysing reagent is followed by treatment with a cross-linking fixing reagent which alters the white cells so that different white cell subpopulations are modified in order that the white cells can be distinguished and classified.
  • the flow analysis instrumentation utilizes radio frequency (RF) current as well as direct current (DC) field excitation for generating the measurement data.
  • RF radio frequency
  • DC direct current
  • optical detection is utilized without as well as with DC field excitation.
  • the desired parameters can be measured directly, or by a mathematical calculation from the direct measurements.
  • FIGURES 1 through 5 are derived from cytograms obtained from normal blood samples.
  • a cytogram is produced by a plurality of points or dots, wherein each dot represents a single cell, and the location of the dot is given by coordinates which are proportional to selected cell parameters; for example, the right angle and forward light ' scatter intensities produced by the cell in the instrument.
  • four clusters of dots or cells are formed, and their areas are encircled in FIGURES 1 through 5 and all of which are identified as leukocytes, namely (1) lymphocytes, (2) monocytes, (3) neutrophils and (4) eosinophils, are the four major categories of leukocytes.
  • saponin Because a high concentration of saponin is needed to provide rapid stromatolyzing of the red cells, it must be used with a rapidly active cross-linking fixing reagent in order to protect the leukocytes against damage. Slow acting monoaldehydes, such as formaldehyde, are ineffective for preserving the leukocytes in this reaction. When saponin alone is added in amounts just barely sufficient to stromatolyse the red blood cells, it requires much too long, about twenty minutes. When used in excess, saponin is a rapid lytic agent, but will severely damage the leukocytes soon after stromatolysing the red blood cells.
  • the formulations can be adjusted to take into account certain general considerations. Since commercial saponin is not a pure material, adjustments in the concentration may be needed for various lots of saponin powder supplied. Within limits, it is possible to change the concentration of saponin added to a given blood sample, if a compensating change in volume of the saponin reagent is also made.
  • the saponin concentration in the lytic diluent ordinarily is within the range of 0.15% to 0.40% w/v.
  • the lytic reagent can contain a higher concentration of saponin, approximately 0.30% to 4.0%, and the alkaline buffer is best included in the prediluent.
  • Preservatives can be added to the lytic diluent and the lytic reagent to inhibit the growth of microorganisms.
  • Water soluble preservatives such as methyl paraben, propyl paraben, formaldehyde, acetaldehyde, dimethylolurea, 2-pyridinethiol-l-oxide, sorbic acid, and potassium sorbate can be used.
  • Additives to the lysing reagent substantially can enhance the separation of the four main white blood cell clusters.
  • the use ' of 2-phenoxyethanol in a range of 0.3 to 0.8% v/v gives enhanced histograms with most blood samples.
  • Other related compounds such as l-phenyl-2-propanol, 2-phenyl-l-propanol, 3-phenoxy-l-propanol and 3-phenyl-l-propanol in the same concentration range result in a similar improvement to the white blood cell histogram.
  • These additives also can include polyhydroxy compounds such as glucose, lactose, and sucrose, in the concentration range of 2 to 8%. Mixtures of more than one additive will give at times improved results.
  • the saponin lytic diluent is formulated for the parameters to be measured.
  • the saponin concentration is in the range of 0.15 to 0.25% w/v
  • the sodium chloride is in 'the range of 0.2 to 0.6% w/v
  • a fluorescent dye is included as is discussed below.
  • cyanine dyes which contain sulfonic acid side chains and which suitably fluoresce by excitation in the blue to green range 488 to 540 nm, or by excitation with red light of 630 to 640 nm, and which are sufficiently stable in aqueous solution.
  • examples of such dyes are:
  • Di S C 3 S0 3 -(5) These are usable with a He/Ne laser at 633 nm light.
  • Thiazine dyes for example, sulforhodami ⁇ e B.
  • Glutaraldehyde is the preferred cross-linking fixing reagent.
  • Other cross-linking dialdehydes include glyoxal, malonaldehyde, and the like. Unsaturated monoaldehydes, such as acrolein or methacrolein, likewise can be used. It is known that acrolein under certain conditions is a bifunctional cross-linking agent.
  • the preferred cross-linking reagent which is added to the lysed blood sample contains glutaraldehyde in the concentration range of 0.5 to 4.0% v/v.
  • This solution also contains sodium chloride in the range of 0.2 to 0.6% w/v and a buffer is included to maintain the pH between 7.0 and 8.0.
  • the buffer can be sodium bicarbonate, phosphates, or Good's buffers, such as morpholinopropanesulfonic acid. If the pH of the reagent system is substantially below 7, there is a tendency for the lysed and fixed blood solution to gel. The pH is maintained within the range of 7.0 to 8.0 throughout the procedures. The osmolality generally falls within the range of 150 to 350 mOs/L.
  • a sodium chloride solution which can contain potassium ferricyanide and potassium cyanide is needed to bring the concentration of cells to a suitable level for counting.
  • the conductivity of the lysed and fixed blood solution is adjusted to be equal to that of the sheath fluid if a focused aperture is used.
  • Potassium ferricyanide and potassium cyanide can be incorporated to convert the oxyhemoglobin to a suitable chromagen, and are incorporated in a range of 0.05 to 5% w/v and 0.01 to 0.2% w/v, respectively.
  • Heating of the lysed and fixed blood solution often is useful to accelerate the modification of the leukocytes and rapidly give stable clusters for flow analysis. Heating to 70 to 75°C for about 10 seconds gives the best results.
  • it is convenient to immerse the blood solution, contained in a glass test tube, into a 70 to 75 ⁇ C water bath with swirling to achieve heating.
  • heating can be accomplished with a coiled electrical resistance wire around the sample chamber, or by other suitable means.
  • Cyanine dye 1 x 10 ⁇ 5 M Di I-C 3 S0 3 ⁇ (3) adjust pH to 7.2 with sodium hydroxide.
  • Fixing reagent 0.5% v/v glutaraldehyde 1.5% w/v sodium bicarbonate
  • To 0.6 ml of the lytic diluent in a test tube is added 50 uL of EDTA anticoagulated whole blood with swirling.
  • 2.0 ml of the fixing reagent is added with swirling.
  • the mixture is heated in a 70°C water bath for fifteen seconds with swirling.
  • the preparation is analyzed on EPICS V Flow Cytometric System using 514 nm He/Ar laser light at 200 mwatt.
  • a 540 nm interference filter and a 570 nm long pass filter are placed in the fluorescence channel.
  • the white blood cell histogram is collected as fluorescence vs light scatter (2° to 20°), as shown in FIGURE 1, or as fluorescence/light scatter vs light scatter.
  • Example 2 White blood cell histogram by fluorescence and light scatter using a COULTER TPS-1 equipped with 30 mwatt HeNe laser at 633 nm. FORMULATIONS Lytic diluent 0.2% w/v saponin
  • the cytogram is collected as fluorescence vs light scatter, as illustrated in FIGURE 2, and shows four distinct clusters.
  • the lymphocytes appear at low fluorescence-low light scatter; the monocytes at intermediate fluorescence-intermediate light scatter; the neutrophils appear at intermediate fluorescence-high light scatter; and the eosinophils at high fluorescence-high light scatter.
  • Example 3 White blood cell histogram by light scatter vs DC volume using a square hole flow cytometer. FORMULATIONS Lytic diluent 0.16% w/v saponin
  • the preparation is analyzed by a COULTER type of flow cytometer containing a flow cell consisting of a COULTER aperture with a square cross section, allowing electro-optical measurements using a -1 mwatt HeNe laser.
  • Forward light scatter, DC volume and RF size can be determined simultaneously with this device.
  • a mask is used to block the narrow forward angle scattering, allowing light scatter to be collected in an approximate range of 10° to 20°, or 10" to 15°.
  • White cell histograms are collected as light scatter vs DC volume, showing four distinct clusters, as illustrated in FIGURE 3.
  • Lymphocytes are seen at low light scatter-low volume; monocytes are seen at low light scatter-high volume; neutrophils are seen at intermediate light scatter-intermediate volume and eosinophils are present at high light scatter-intermediate volume.
  • the square hole flow cytometer and its uses are described more fully by R.A. Thomas, T.A. Yopp, B.D. Watson, D.H.K. Hindman, B.F. Cameron, S.B. Leif, B.C. Leif, L. Roque and W. Britt in the Journal of Histochemistry and Cytochemistry, Vol. 25, No. 77, pp. 827-835 (1977). Cytograms also can be collected as light scatter/DC volume vs DC volume.
  • Example 4 White blood cell histogram by opacity (RF size/DC volume) and DC volume using the square hole flow cell, HeNe laser. FORMULATIONS
  • Example 5 The preparation is analyzed by the instrument described in Example 3, measuring opacity vs DC volume. Four distinct clusters of white blood cells are found, as shown in FIGURE 4. Lymphocytes appear at low to high opacity-low volume; monocytes are seen at low opacity-high volume; neutrophils are seen at intermediate opacity-high volume and eosinophils are seen at high opacity-high volume. This procedure also gives good cytograms in the 10° to 20" light scatter vs DC volume parameters. The RF, DC and light scatter parameters can be collected simultaneously and used together by forming ratios, or by selective gating to give improved cluster definition. Example 5
  • the sample is diluted 10 times with isotonic diluent. No correction for conductivity is needed. Determination of the opacity (RF size/DC volume) vs DC volume gives four leukocyte clusters, as shown in FIGURE 5. The lymphocytes appear at low to high opacity-low volume; monocytes and neutrophils are seen at intermediate opacity and high volume, as clearly separated populations; and eosinophils are located at high opacity and high volume.
  • Example 6 White blood cell histogram by opacity (RF size/DC vol) vs DC volume parameters using a COULTER COUNTER Model S Plus, with mixing chamber and focused aperture adaptions.
  • the instrument aspirates a whole blood sample anticoagulated with EDTA into the sampling loop, where 28 fL of blood is segmented.
  • a 170 uL portion of the lytic reagent is delivered into the sample chamber, followed promptly by the whole blood sample carried by 150 uL of the prediluent.
  • Mixing of the sample chamber by nutation is started and 100 uL of the lytic reagent is added immediately. After seven seconds lysing time a 250 uL portion of the fixing reagent is added. Heating is initiated immediately, bringing the sample to 70°C in about seven seconds.
  • the mixing is stopped, and the sample is fed into a focused flow cell for measurement of the DC volume and RF size parameters.
  • the bottom drain is opened and the excess sample is removed from the sample chamber, which is further rinsed in order to reduce "carry over", and to cool the sample chamber in preparation for the next sample.

Abstract

A reagent system, including saponin in a lysing reagent and a rapidly active cross-linking agent such as glutaraldehyde as a fixing reagent, which reproducably affects whole blood to cause the red blood cells to stromatolyze and modifies the leukocytes to generate data to define four distinct clusters for detection and classification by flow analysis instrumentation. The clusters represent the four major leukocyte types found in blood: lymphocytes, monocytes, neutrophils and eosinophils, thus providing a method of leukocyte differential analysis. The parameters used for the leukocyte classification include combinations of two or more of DC (Coulter) volume, high frequency (RF) size, opacity (RF size/DC volume), light scatter at various angular ranges, and fluorescence at various wavelengths of illumination.

Description

METHOD AND REAGENT SYSTEM FOR FOUR-POPULATION DIFFERENTIAL ETERMINATION OF LEUKOCYTES
Many methods have been used for removing the red blood cells from whole blood so that the leukocytes can be studied by flow techniques. Physical separation by sedimentation or centrifugation or density gradients, aggregation of red blood cells and other physical techniques are useful for research purposes, but are too slow and difficult for automated clinical analysis of leukocytes. Quaternary ammonium salt detergents are very efficient lytic agents, but have been found to be too damaging to the leukocytes, resulting at best, in only three clusters of leukocytes, by DC volume analysis, representing the lymphocytes, monocytes and granulocytes.
Kim, U. S. Patent 4,099,917, 1978, describes a method of sensitizing red blood cells with a non-ionic detergent, adding a formaldehyde fixative, and incubating the blood at 58°C, to lyse the red blood cells selectively, leaving leukocytes and platelets intact for light scatter measurements. This process is rather slow, about three minutes, and may be sufficient to make the red blood cells transparent toward optical measurements, but not towards electronic measurements, which require more thorough stromatolyzing of the red blood cells. The same is true of other lytic procedures, such as hypotonic lysis, ammonium chloride lysis, and ethylene or propylene glycol treatment which render the red blood cells transparent towards optical, i.e. light scatter, fluorescence measurements, but not towards electronic, i.e. DC volume and R.F. volume parameters.
The natural product known as saponin has long been used as a red blood cell lytic agent. Saponin is chemically defined as a class of glycosides of various mono- or polysaccharides, with steroid or triterpene alcohols. Quillaja saponin is isolated as a natural' product from quillaja tree bark. This saponin has detergent-like properties and hemolyzes red blood cells when used at very low concentrations compared to synthetic ionic and nonionic hemolytic agents. However, the chemical treatment, structure and purity of commercial quillaja saponin is not generally specified or tested, and the material does vary from lot to lot.
The activity of saponin is more selective towards red blood cells than are the quaternary ammonium salt detergents. Unfortunately, by employing lysing procedures known heretofore, it has not been possible to obtain leukocytes free from red blood cells, without doing some concomitant damage to the leukocytes.
Several reports describe the use of saponin with a second reagent which retards the leukocyte damage. Hughes-Jones, J. Clin. Path.,
Vol. 27, page 623 (1974), reported a treatment of diluted whole blood with a saponin solution, followed in three minutes by treatment with serum which quenches the saponin activity. A direct current volume analysis was done on the leukocytes. Ornstein, Blood Cells, Vol. 25, page 57 (1976), used a solution of saponin, formaldehyde and other components, for twenty seconds followed by enzyme staining for detecting various leukocyte types. Humphries et al, Ser. Haemat. Vol. V-2, page 142 (1972), treat diluted whole blood with saponin, followed in thirty seconds with dilution in cold phosphate buffered saline to quench the lytic action. Other reports are Ladinsky, Cancer Res.,
Vol. 27, page 1689 (1967), and Van Dilla, Proc. Soc. Exp. Biol. (NY), Vol. 125, page 367 (1967).
Commercial equipment employing the teachings of U. S. Patents 2,656,508; and 3,259,842 are known under the trademark COULTER COUNTER®, and the principle of their operation is commonly known as the Coulter principle.
According to the Coulter principle, first patented in U. S. Patent 2,656,508, 1953, when a particle of microscopic size is passed through an electrical field of small dimensions of an order approaching those of a particle, there will be a momentary change in the electric impedence. If the electrical field is excited by a direct (DC) or low frequency current, the electrical change is closely proportional to the volume of the particle. In commerical apparatus, the changes are detected by some suitable means and used to operate counters and analyzers. The analyzers associated with such apparatus classify and size particles into populations based upon particle volume and record the data obtained.
The invention was materially expanded in U. S. Patent 3,502,974, Coulter et al, 1970, using radio frequency (RF) current in addition to DC current field excitation to provide not only DC volume information concerning the particle studied, but also information due to the composition and nature of the material constituting the particle. This patent discloses apparatus capable of distinguishing between particles of identical size, but of different material. By generating the particle sensing field by means of both a low frequency or direct current (DC) and radio frequency (RF) current excitation, two or more interrelated output signals can be derived from the passage of a single particle through the electrical field. This is due to the fact that, although the subject particles are nearly always insulators with respect to low frequency or direct current fields, they are capable of carrying or impeding radio frequency current differently from the surrounding electrolyte. This may be due to differences in the dielectric constant in the case of homogeneous particles, or to the sac-like structure in the case of blood cells which have, enclosed in an extremely thin membrane, contents having conductivities different from the electrolyte. Thus, while all the DC current goes around a blood cell, some of the RF current will go through it. The ease with which the RF current will go through a particle is a measure of what is termed its "electrical transparency", or simply "transparency", in analogy with light transmission; whereas, a particle's ability to impede RF current is termed its "opacity". In later publications, "opacity" is defined as the RF impedence divided by the DC impedence.
The relative electrical opacity of a particle becomes an identifying feature of the particle- contents, and hence, its particle type for classification purposes. To the extent that different types of particles each possess a different opacity, the difference between them is detectable. However, significantly different particles' can possess substantially the same opacity and such particles cannot be classified effectively in this manner. In U. S. Patent 3,836,849, 1974, Coulter et al taught that it is possible to change selectively the opacity of particle types by treatment of the particles, so that detectable differences result.
Although red blood cells and white blood cells nominally have different sizes, their size ranges tend to overlap, or at least under certain conditions of health could overlap. Moreover the opacities of these two types of blood cells may also overlap.
U. S. Patent 3,741,875, Ansley et al, June, 1973, describes a process for obtaining a differential white blood cell count. A cytological fixing agent, which is a monoaldehyde such as formaldehyde, is added to a blood sample. A hemolyzing agent then is added after the fixation step to cause the red blood cells to release their hemoglobin content into solution. Addition of a specific cytochemical substrate, chromogenic precipitating coupling reagent, and pH buffer causes deposition of an insoluble dye in a specific type of cell containing an immobilized enzyme. The solution containing the dyed blood cells then is passed through a photometric counter. Using different specific substrates for different enzymes contained in specific kinds of cells, absolute and relative counts of the different kinds of cells are obtained. The cytological fixing solution utilized only a monoaldehyde. Dialdehydes are stated to be unsuitable, since they cross-link and produce extracellular precipitates.
Starting with whole blood, it is necessary to hemolyze the red blood cells, since there is danger that coincident passage of two or more red cells through a photometric counting station could be mistaken for dyed white blood cells or abnormal cells. A preferred way to solve the problem is to hemolyze the red blood cells by addition of a reagent to the suspension of cells to cause the red blood cells to rupture and release their hemoglobin content into the solution.
Ledis et al, U. S. Patent 4,286,963, 1981, teaches a method for two-volume analysis of leukocytes using a COULTER COUNTER analyzer which employs only DC field excitation instrumentation and quaternary ammonium salts as lysing agents. Ledis et al, U. S. Patent 4,485,175, to Coulter Electronics, Inc. concerns a method and reagent system for three-volume differential determination of lymphocyte, monocyte, and granulocyte populations of leukocytes, using quaternary ammonium salts as lysing agents and the COULTER COUNTER Model S Plus automated blood counter, which instrument employs only direct current field excitation. Previous methods of flow analysis of leukocytes using DC volume, or light scatter at various angles have shown only three clusters of leukocytes, corresponding to lymphocytes, monocytes, and granulocytes including neutrophils and eosinophils. The eosinophils have been observed as a distinct cluster by using special fluorescence techniques.
Other dye compositions for differential analysis of white blood cells include a hypotonic aqueous solution of a metachromatic fluorochrome dye such as acridine orange, Adams, U. S. Patent 3,883,247. The white cell analysis is made by suspending a sample of fresh blood in the dye solution, subjecting the suspension, before dye uptake equilibrium is reached, to radiation from a light source, e.g. radiation from a blue laser, having a wave length within the range of absorption of the dye, and distinguishing the white cells from other blood particles by detecting fluorescences, e.g. green vs. red fluorescences.
Fluorescent dyes suitable for specifically dyeing eosinophil granules are the anilino or toluidino naphthalene sulfonic acids and their alkyl, alkoxy or halogen substituted derivatives. The development of instrumentation and fluorochromes for automated multiparameter analysis of cells is further described by R.C. Leif et al. in Clinical Chemistry, Vol. 23, pp 1492-98 (1977).
Eosinophils have been observed also by enzyme staining such as by the Technicon Hemalog D and H6000 instruments, (Ansley and Ornstein, Adv. Automated Anal., Vol. 1 437 (1971), Kaplow, Macrophages and Lymphocytes, Part A, 211 Plenum (1980).
The detection of populations of particular leukocytes, and' the concurrent relationship of these populations to one another in a human blood sample is important in medical research and for the diagnosis of certain human diseases. Such data are useful as a screening tool for calling attention to abnormal leukocyte ratios. Abnormal situations identified by this method give information of diagnostic significance and alert the technologist to the need for further study.
This invention concerns a reagent system and a method for classifying and counting at least four populations of leukocytes using flow analysis instrumentation. More particularly, the present invention relates to a multicomponent reagent system for rapidly eliminating the red blood cells in a sample of whole blood and for maintaining and modifying leukocytes in a manner suitable for flow analysis classification into four major categories which have been identified as (1) lymphocytes, (2) monocytes, (3) neutrophils and (4) eosinophils.
The reagent system comprises aqueous solutions of: (A) a lytic diluent containing saponin; or a blood diluent which is followed by a lytic reagent containing saponin; (B) a fixing reagent containing a cross-linking compound such as glutar ldehyde, each of these two components being maintained at a predetermined concentration, pH and osmolality. This method is rapid, reliable and effective for normal and abnormal bloods and blood control material. Preservatives can be added to inhibit the growth of microorganisms. Other additives also can be included. A fluorescent dye is included in the lytic diluent when studies make a determination by fluorescence.
The lysed and fixed blood sample is brought to the proper concentration for flow analysis using a diluent which also can contain potassium ferricyanide and potassium cyanide for converting the hemoglobin to a chromagen suitable for hemoglobinometry.
This invention relates also to a method for discriminating of major categories of leukocytes by flow instrumentation which measures two or more of the primary parameters of DC volume, RF size, fluorescence and light scatter, and also provides histograms based on selected mathematical combinations of the primary parameters.
By way of example, illustrative embodiments of the invention now will be described with reference to the accompanying drawings in which:
FIGURES 1 through 5 illustrate the leukocyte subpopulation identification accomplished by the method of this invention. In the diagrams of the FIGURES, the encircled areas represent cell populations or cell clusters that are generated by accumulation of data points, in which each data point is determined by coordinates which are proportional to certain cell parameters. This form of diagram is known as a cytogram. The hereinafter presented Examples explain each FIGURE.
The purpose of this invention is to provide a method for the rapid lysing of the red blood cells in whole blood in a manner that preserves and/or modifies the leukocytes, so that they can be distinguished or classified into subpopulations. In the present invention, whole blood is treated with a lysing reagent. This lysing reagent has two forms: (D a lytic diluent containing saponin, which simultaneously functions to dilute the whole blood sample and stromatolyse its red blood cells; or (2) a two part system comprised of non-lytic blood diluent followed by a lytic reagent containing saponin. The lysing reagent is followed by treatment with a cross-linking fixing reagent which alters the white cells so that different white cell subpopulations are modified in order that the white cells can be distinguished and classified. In some embodiments, the flow analysis instrumentation utilizes radio frequency (RF) current as well as direct current (DC) field excitation for generating the measurement data. In other embodiments, optical detection is utilized without as well as with DC field excitation. The desired parameters can be measured directly, or by a mathematical calculation from the direct measurements.
FIGURES 1 through 5 are derived from cytograms obtained from normal blood samples. A cytogram is produced by a plurality of points or dots, wherein each dot represents a single cell, and the location of the dot is given by coordinates which are proportional to selected cell parameters; for example, the right angle and forward light' scatter intensities produced by the cell in the instrument. In this manner, four clusters of dots or cells are formed, and their areas are encircled in FIGURES 1 through 5 and all of which are identified as leukocytes, namely (1) lymphocytes, (2) monocytes, (3) neutrophils and (4) eosinophils, are the four major categories of leukocytes.
Because a high concentration of saponin is needed to provide rapid stromatolyzing of the red cells, it must be used with a rapidly active cross-linking fixing reagent in order to protect the leukocytes against damage. Slow acting monoaldehydes, such as formaldehyde, are ineffective for preserving the leukocytes in this reaction. When saponin alone is added in amounts just barely sufficient to stromatolyse the red blood cells, it requires much too long, about twenty minutes. When used in excess, saponin is a rapid lytic agent, but will severely damage the leukocytes soon after stromatolysing the red blood cells.
In the examples which follow, the formulations can be adjusted to take into account certain general considerations. Since commercial saponin is not a pure material, adjustments in the concentration may be needed for various lots of saponin powder supplied. Within limits, it is possible to change the concentration of saponin added to a given blood sample, if a compensating change in volume of the saponin reagent is also made. The saponin concentration in the lytic diluent ordinarily is within the range of 0.15% to 0.40% w/v.
When the blood sample is prediluted with a non-lytic diluent, the lytic reagent can contain a higher concentration of saponin, approximately 0.30% to 4.0%, and the alkaline buffer is best included in the prediluent.
Preservatives can be added to the lytic diluent and the lytic reagent to inhibit the growth of microorganisms. Water soluble preservatives, such as methyl paraben, propyl paraben, formaldehyde, acetaldehyde, dimethylolurea, 2-pyridinethiol-l-oxide, sorbic acid, and potassium sorbate can be used.
Additives to the lysing reagent substantially can enhance the separation of the four main white blood cell clusters. The use' of 2-phenoxyethanol in a range of 0.3 to 0.8% v/v gives enhanced histograms with most blood samples. Other related compounds, such as l-phenyl-2-propanol, 2-phenyl-l-propanol, 3-phenoxy-l-propanol and 3-phenyl-l-propanol in the same concentration range result in a similar improvement to the white blood cell histogram. These additives also can include polyhydroxy compounds such as glucose, lactose, and sucrose, in the concentration range of 2 to 8%. Mixtures of more than one additive will give at times improved results. The saponin lytic diluent is formulated for the parameters to be measured. For determinations of fluorescence, the saponin concentration is in the range of 0.15 to 0.25% w/v, the sodium chloride is in 'the range of 0.2 to 0.6% w/v, and a fluorescent dye is included as is discussed below.
Generally, cyanine dyes are employed which contain sulfonic acid side chains and which suitably fluoresce by excitation in the blue to green range 488 to 540 nm, or by excitation with red light of 630 to 640 nm, and which are sufficiently stable in aqueous solution. Examples of such dyes are:
A. Cyanine dyes at a concentration of about 1 x 10~° M to 5 x 10"5 M:
Di I C3S03-(3)
Di 0 C3S03-(3) Di S C3S03-(3)
0C S03 - TBA-Cχ (4) and many others. These are usable with a He/Ar laser at 488 or 514 nm light.
B. Cyanine dyes at an approximate concentration 1 x 10"^ M: Di I C3S03-(5)
Di S C3S03-(5) These are usable with a He/Ne laser at 633 nm light.
C. Thiazine dyes, for example, sulforhodamiπe B.
The abbreviations for the dyes are those used by Alan Waggoner, Biochem 13 3315 (1974). He/Ar means Helium/Argon; whereas, He/Ne means Helium/Neon.
Glutaraldehyde is the preferred cross-linking fixing reagent. Other cross-linking dialdehydes include glyoxal, malonaldehyde, and the like. Unsaturated monoaldehydes, such as acrolein or methacrolein, likewise can be used. It is known that acrolein under certain conditions is a bifunctional cross-linking agent.
The preferred cross-linking reagent which is added to the lysed blood sample contains glutaraldehyde in the concentration range of 0.5 to 4.0% v/v. This solution also contains sodium chloride in the range of 0.2 to 0.6% w/v and a buffer is included to maintain the pH between 7.0 and 8.0. The buffer can be sodium bicarbonate, phosphates, or Good's buffers, such as morpholinopropanesulfonic acid. If the pH of the reagent system is substantially below 7, there is a tendency for the lysed and fixed blood solution to gel. The pH is maintained within the range of 7.0 to 8.0 throughout the procedures. The osmolality generally falls within the range of 150 to 350 mOs/L.
A sodium chloride solution, which can contain potassium ferricyanide and potassium cyanide is needed to bring the concentration of cells to a suitable level for counting. The conductivity of the lysed and fixed blood solution is adjusted to be equal to that of the sheath fluid if a focused aperture is used. Potassium ferricyanide and potassium cyanide can be incorporated to convert the oxyhemoglobin to a suitable chromagen, and are incorporated in a range of 0.05 to 5% w/v and 0.01 to 0.2% w/v, respectively.
Heating of the lysed and fixed blood solution often is useful to accelerate the modification of the leukocytes and rapidly give stable clusters for flow analysis. Heating to 70 to 75°C for about 10 seconds gives the best results. In manual preparations, it is convenient to immerse the blood solution, contained in a glass test tube, into a 70 to 75βC water bath with swirling to achieve heating. In an automated system, heating can be accomplished with a coiled electrical resistance wire around the sample chamber, or by other suitable means. Example 1
White blood cell cytogram by fluorescence and light scatter using a COULTER® EPICS® V Flow Cytometer.
FORMULATIONS Lytic diluent 0.2% w/v saponin 0.6% w/v sodium chloride
1.0% Hepes Buffer
Cyanine dye 1 x 10~5 M Di I-C3S03~(3) adjust pH to 7.2 with sodium hydroxide. Fixing reagent 0.5% v/v glutaraldehyde 1.5% w/v sodium bicarbonate To 0.6 ml of the lytic diluent in a test tube is added 50 uL of EDTA anticoagulated whole blood with swirling. As soon as the blood solution clears in about five to ten seconds, 2.0 ml of the fixing reagent is added with swirling. After fifteen seconds the mixture is heated in a 70°C water bath for fifteen seconds with swirling.
The preparation is analyzed on EPICS V Flow Cytometric System using 514 nm He/Ar laser light at 200 mwatt. A 540 nm interference filter and a 570 nm long pass filter are placed in the fluorescence channel. The white blood cell histogram is collected as fluorescence vs light scatter (2° to 20°), as shown in FIGURE 1, or as fluorescence/light scatter vs light scatter.
Four distinct clusters of white blood cells are observed. Sorting of cells in each of these clusters and microscopic examination shows that the cells are lymphocytes at low light scatter light scatter-low fluorescence; monocytes at intermediate light scatter-intermediate fluorescence; neutrophils at a high light scatter-high fluorescence; and eosinophils at high light scatter-very high fluorescence. The dye strongly stains the eosoniphilic granules of eosinophils and neutrophils, and weakly stains the cytoplasm of all the white blood cells.
A fifth cluster, not illustrated, sometimes is observed at low light scatter-intermediate fluorescence.
Example 2 White blood cell histogram by fluorescence and light scatter using a COULTER TPS-1 equipped with 30 mwatt HeNe laser at 633 nm. FORMULATIONS Lytic diluent 0.2% w/v saponin
0.5% w/v sodium sulfate 3.0% w/v sucrose cyanine dye 1 x 10"5 M DiIC S03~(5) Fixing reagent 1.0% v/v glutaraldehyde
0.8% w/v sodium chloride 0.5% w/v sodium bicarbonate To 1.0 ml of the lytic diluent in a test tube is added with swirling 0.1 ml of whole blood anticoagulated with EDTA. As soon as the blood solution clears in about five to seven seconds, 2.0 ml of the fixing reagent is added with swirling. After fifteen seconds, the mixture is heated in a 60°C to 70°C water bath with swirling for thirty seconds. The preparation is analyzed by a COULTER TPS-1 Flow Cytometer equipped with a 30 mwatt HeNe laser. Forward light scatter is measured in an angular range of 2°to 20". Fluorescence is measured using a 665 nm long pass filter. The cytogram is collected as fluorescence vs light scatter, as illustrated in FIGURE 2, and shows four distinct clusters. The lymphocytes appear at low fluorescence-low light scatter; the monocytes at intermediate fluorescence-intermediate light scatter; the neutrophils appear at intermediate fluorescence-high light scatter; and the eosinophils at high fluorescence-high light scatter.
Example 3 White blood cell histogram by light scatter vs DC volume using a square hole flow cytometer. FORMULATIONS Lytic diluent 0.16% w/v saponin
0.2% w/v sodium sulfate 4.0% w/v sucrose Fixing reagent 1.5% v/v glutaraldehyde
0.1% w/v sodium bicarbonate 0.4% w/v sodium chloride
To 1.5 ml of the lytic diluent in a test tube is added with swirling 0.1 ml whole blood anticoagulated with EDTA. As soon as the blood solution clears, in approximately five to seven seconds, 2.0 ml of the fixing reagent is added with swirling. The solution is brought to isoconductivity with respect to the sheath fluid by addition of a suitable volume of 1.6% w/v sodium chloride to the solution.
The preparation is analyzed by a COULTER type of flow cytometer containing a flow cell consisting of a COULTER aperture with a square cross section, allowing electro-optical measurements using a -1 mwatt HeNe laser. Forward light scatter, DC volume and RF size can be determined simultaneously with this device. A mask is used to block the narrow forward angle scattering, allowing light scatter to be collected in an approximate range of 10° to 20°, or 10" to 15°. White cell histograms are collected as light scatter vs DC volume, showing four distinct clusters, as illustrated in FIGURE 3. Lymphocytes are seen at low light scatter-low volume; monocytes are seen at low light scatter-high volume; neutrophils are seen at intermediate light scatter-intermediate volume and eosinophils are present at high light scatter-intermediate volume. The square hole flow cytometer and its uses are described more fully by R.A. Thomas, T.A. Yopp, B.D. Watson, D.H.K. Hindman, B.F. Cameron, S.B. Leif, B.C. Leif, L. Roque and W. Britt in the Journal of Histochemistry and Cytochemistry, Vol. 25, No. 77, pp. 827-835 (1977). Cytograms also can be collected as light scatter/DC volume vs DC volume.
Example 4 White blood cell histogram by opacity (RF size/DC volume) and DC volume using the square hole flow cell, HeNe laser. FORMULATIONS
Lytic diluent 0.4% w/v saponin
0.2% w/v sodium chloride 0.5% v/v 2-phenoxyethanol 0.03% w/v methyl paraben preservative Fixing reagent 2.0% v/v glutaraldehyde
0.4% w/v sodium chloride 0.1% w/v sodium bicarbonate To 0.6 ml of lytic diluent in a test tube is added with swirling 0.10 ml whole blood anticoagulated with EDTA. After ten seconds, 1.0 of fixing reagent is added with swirling. The mixture is heated in a 70°C water bath for fifteen seconds. The solution is brought to isoconductivity with the sheath fluid by addition of a suitable volume of 1.6% w/v sodium chloride.
The preparation is analyzed by the instrument described in Example 3, measuring opacity vs DC volume. Four distinct clusters of white blood cells are found, as shown in FIGURE 4. Lymphocytes appear at low to high opacity-low volume; monocytes are seen at low opacity-high volume; neutrophils are seen at intermediate opacity-high volume and eosinophils are seen at high opacity-high volume. This procedure also gives good cytograms in the 10° to 20" light scatter vs DC volume parameters. The RF, DC and light scatter parameters can be collected simultaneously and used together by forming ratios, or by selective gating to give improved cluster definition. Example 5
White blood cell histogram by opacity (RF size/DC volume), vs DC volume using the focused square hole flow cell or an unfocused standard COULTER aperture. Demonstration of the predilution method. FORMULATIONS
Prediluent 0.3% w/v sodium chloride
0.1% w/v sodium bicarbonate Lytic reagent 2.5% w/v saponin
0.4% w/v sodium chloride 0.1% w/v sorbic acid (preservative)
Fixing reagent 2.0% v/v glutaraldehyde
0.4% w/v sodium chloride To 1.0 ml of the prediluent is added with swirling 0.10 ml of whole blood, anticoagulated with EDTA, followed by 0.10 ml of the lytic reagent. After ten seconds, 1.0 ml of the fixing reagent is added with swirling, and the sample is heated in a 70°C water bath for fifteen seconds. For use with a focused square hole aperture, the sample is diluted 1 : 1 with 0.9% w/v of sodium chloride and the conductivity is adjusted to be equal to that of the sheath fluid by suitable addition of 2.0% w/v of sodium chloride.
For use with an unfocused aperture in an aperture bath, the sample is diluted 10 times with isotonic diluent. No correction for conductivity is needed. Determination of the opacity (RF size/DC volume) vs DC volume gives four leukocyte clusters, as shown in FIGURE 5. The lymphocytes appear at low to high opacity-low volume; monocytes and neutrophils are seen at intermediate opacity and high volume, as clearly separated populations; and eosinophils are located at high opacity and high volume.
Example 6 White blood cell histogram by opacity (RF size/DC vol) vs DC volume parameters using a COULTER COUNTER Model S Plus, with mixing chamber and focused aperture adaptions.
FORMULATIONS Prediluent 0.3% w/v sodium chloride 0.4% w/v sodium bicarbonate
Lytic reagent 0.35% w/v saponin
0.4% w/v sodium chloride 0.1% w/v acetaldehyde preservative Fixing reagent 3.0% w/v glutaraldehyde 0.4% w/v sodium chloride
Diluent 4.0% w/v sodium chloride
The instrument aspirates a whole blood sample anticoagulated with EDTA into the sampling loop, where 28 fL of blood is segmented. A 170 uL portion of the lytic reagent is delivered into the sample chamber, followed promptly by the whole blood sample carried by 150 uL of the prediluent. Mixing of the sample chamber by nutation is started and 100 uL of the lytic reagent is added immediately. After seven seconds lysing time a 250 uL portion of the fixing reagent is added. Heating is initiated immediately, bringing the sample to 70°C in about seven seconds. After seven more seconds at this temperature 170 uL of the diluent is added for conductivity adjustment, the mixing is stopped, and the sample is fed into a focused flow cell for measurement of the DC volume and RF size parameters. After sampling, the bottom drain is opened and the excess sample is removed from the sample chamber, which is further rinsed in order to reduce "carry over", and to cool the sample chamber in preparation for the next sample.
Measurement of the DC volume and RF size parameters allow the discrimination of four leukocyte populations as described in the former examples and as shown in FIGURE 5. While in the foregoing specification, a detailed description of the invention has been set down for the purpose of illustration, many variations in the details herein given may be made by those skilled in the art without departing from the spirit and scope of the invention.

Claims

WHAT WE CLAIM IS:
1. A multicomponent reagent system for stromatolyzing the red blood cells in a sample of whole blood and for maintaining and modifying leukocytes in a manner suitable for flow analysis classification into major categories, said reagent system characterized by an aqueous solutions of:
(A) a lysing reagent selected from the group consisting of a lytic diluent containing saponin, and a prediluent system followed by a lytic reagent containing saponin; and (B) a fixing reagent containing a cross-linking compound; each of said components (A) and (B) being maintained at a predetermined concentration, pH and osmolality.
2. The reagent system of claim 1 characterized in which said saponin is quillaja saponin.
3. The reagent system of claims 1 or 2 characterized wherein said lytic diluent provides a saponin concentration in the range of 0.15 to 0.4% w/v in the presence of whole blood and contains an amount of saponin in the range of 0.0020 to 0.0025 grams of saponin for each 100 uL of whole blood.
4. The reagent system of any one of claims 1 to 3 characterized wherein said lytic diluent consists of 0.15% to 0.40% w/v of saponin, 0.20% to 0.60% w/v of sodium chloride or sodium sulfate, and 0.01% to 0.10% w/v of sorbic acid, the volume of lytic reagent being 6 to 15 times the volume of the whole blood sample.
5. The reagent system of any one of claims 1 to 4 characterized wherein said lysing reagent contains alkali metal salts in a concentration range of 0.2% to 0.6% w/v.
6. The reagent system of any one of claims 1, 2 or 5 characterized wherein said lytic reagent contains saponin in a concentration range of 0.30% to 4.0% w/v.
7. The reagent system of claim 6 characterized wherein the volume of lytic reagent added to the prediluted blood is approximately 1 to 10 times that of the whole blood sample, and then adjusted according to a predetermined concentration of saponin in the lytic reagent, such that the amount of saponin present in the lysing reagent is 0.0020 to 0.0025 grams per 100 uL of whole blood, and a saponin concentration in the lysing reagent of 0.15 to 0.40% w/v.
8. The reagent system of any one of claims 1 to 7 characterized in that there has been added a water soluble preservative which inhibits the growth of microorganisms.
9. The reagent system of claim 8 characterized wherein said preservative is selected from the group consisting of water soluble bacteriocides, fungicides, sorbic acid, potassium sorbate, methyl paraben, propyl paraben, formaldehyde, acetaldehyde, dimethylolurea, 2-pyridinethiol-l-oxide, and sodium azide.
10. The reagent system of any one of claims 1 to 9 characterized by including, for enhancing cell classification, at least one additive selected from the group consisting of a short chain alkanol substituted by phenyl or phenoxy and a polyhydroxy compound.
11. The reagent system of claim 10 characterized wherein said additive is 2-phenoxyethanol.
12. The reagent system of claims 10 or 11 characterized wherein said polyhydroxy compound is sucrose.
13. The reagent system of any one of claims 1 to 12 characterized by including a fluorescent dye.
14. The reagent system of claim 13 characterized wherein said fluorescent dye is a sulfonated cyanine dye or other sulfonated fluorescent dye.
15. The reagent system of any one of claims 1 to 14 characterized wherein an aqueous prediluent is added to said sample of whole blood, said prediluent containing a predetermined concentration of conventional buffers and salts to maintain the pH in the range of 7.0 to 8.0.
16. The reagent system of any one of claims 1 to 15 characterized wherein said cross-linking compound is glutaraldehyde.
17. The reagent system of claim 16 characterized wherein said glutaraldehyde is present in a concentration range of 0.5% to 4.0% w/v.
18. The reagent system of any one of claims 1 to 17 characterized wherein said fixing, reagent consists of glutaraldehyde in a concentration range of 0.5% to 4.0% v/v, an alkali metal chloride or sulfate in a concentration range of 0.2% to 0.8% w/v, and a buffer salt in a concentration range of 0.0% to 2.0% w/v to maintain pH in the range of 7.0 to 8.0, the volume of fixative solution added to the lysed whole blood being in the range of 1 to 5 times that of the yse solution, so that the concentration of glutaraldehyde in the lysed and fixed blood solution is in the range of 0.20% to 2.0% v/v.
19. The reagent system of any one of claims 1 to 18 characterized wherein said major leukocyte categories are: lymphocytes, monocytes, neutrophils and eosinophils.
20. A method for discriminating between major categories of leukocytes by flow analysis instrumentation which comprises: treating a sample of whole blood with a reagent system which stromatolyzes the red blood cells and enables the leukocytes to be classified into major categories, said reagent system characterized by being an aqueous solution of:
(A) a lysing reagent selected from the group consisting of a lytic diluent containing saponin, and a prediluent system containing saponin; and (B) a fixing reagent containing a cross-linking compound; maintaining each of said components (A) and (B) at a predetermined concentration, pH and osmolality; and measuring by said instrumentation at least two of the primary parameters of DC volume, RF size, fluorescence, and light scatter to obtain leukocyte category data.
21. The method of claim 20 characterized by heating said treated blood at an elevated temperature for a few seconds.
22. The method of claim 21 characterized wherein said heating takes place at a temperature of about 60" to 75βC for about 10 to 30 seconds.
23. The method of any one of claims 20 to 22 characterized by generating cytograms by said instrumentation, said cytograms being based on selected mathematical combinations of said primary parameters.
24. The method of claim 20 which further is characterized by correlating the measured parameters of RF size/DC volume vs DC volume.
25. The method of claim 20 which further is characterized by correlating the measured parameters of light scatter vs DC volume.
26. The method of claim 20 which further is characterized by correlating the measured parameters of light scatter/DC volume vs DC volume.
27. The method of claim 20 which further is characterized by correlating the measured parameters of fluorescence vs light scatter.
28. The method of claim 20 which further is characterized by correlating of the measured parameters of fluorescence/light scatter vs light scatter.
29. The method of any one of claims 20 to 28 characterized wherein said major categories of leukocytes are: lymphocytes, monocytes, neutrophils and eosinophils.
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0259834A2 (en) * 1986-09-10 1988-03-16 Toa Medical Electronics Co., Ltd. Method of classifying leucocytes by flow cytometry
WO1988007198A1 (en) * 1987-03-13 1988-09-22 Coulter Electronics, Inc. Multi-part differential analyzing apparatus utilizing light scatter techniques
US4791355A (en) * 1986-10-21 1988-12-13 Coulter Electronics Inc. Particle analyzer for measuring the resistance and reactance of a particle
FR2654744A1 (en) * 1989-11-20 1991-05-24 Abx Sa REAGENT AND METHOD OF USE THEREOF FOR THE AUTOMATIC DETERMINATION IN CYTOMETRY OF FLOWS OF AT LEAST ONE LEUKOCYTE SUB-POPULATION FROM TOTAL BLOOD.
EP0483116A2 (en) * 1986-11-27 1992-04-29 Toa Medical Electronics Co., Ltd. Method of classifying leukocytes by flow cytometry and reagents used in the method
EP0574267A2 (en) * 1992-06-12 1993-12-15 Gen-Probe Incorporated Preparation of nucleic acid from blood
EP0582736A1 (en) * 1992-08-11 1994-02-16 Abbott Laboratories Flow cytometry sheath reagent for elimination of red cells with preservation of nucleated cells
EP0642022A2 (en) * 1993-09-07 1995-03-08 Heinrich Prof. Dr. Patscheke Stabilizing medium for blood
WO1996034291A1 (en) * 1995-04-28 1996-10-31 Coulter International Corp. Quality control method of hematology instruments
DE19520298A1 (en) * 1995-06-02 1996-12-05 Bayer Ag Sorting device for biological cells or viruses
WO1998026284A1 (en) * 1996-12-11 1998-06-18 Nycomed Amersham Plc Selective lysis of cells
CN1040469C (en) * 1992-11-19 1998-10-28 东亚医用电子株式会社 A pretreatment method for blood analysis
EP2278331A1 (en) * 2008-05-02 2011-01-26 ARKRAY, Inc. Leukocyte analysis method and analysis reagent for use in the method
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Families Citing this family (73)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5188935A (en) * 1984-05-31 1993-02-23 Coulter Electronics, Inc. Reagent system and method for identification, enumeration and examination of classes and subclasses of blood leukocytes
CA1255197A (en) * 1984-09-24 1989-06-06 Joseph L. Orlik Leukocyte differentiation composition and method
US5175109A (en) * 1986-09-10 1992-12-29 Toa Medical Electronics Co., Ltd. Reagent for classifying leukocytes by flow cytometry
US5179026A (en) * 1986-11-27 1993-01-12 Toa Medical Electronics Co., Ltd. Method of classifying leukocytes by flow cytometry and reagents used in the method
US5155044A (en) * 1987-03-13 1992-10-13 Coulter Electronics, Inc. Lysing reagent system for isolation, identification and/or analysis of leukocytes from whole blood samples
US6159740A (en) * 1987-03-13 2000-12-12 Coulter Corporation Method and apparatus for screening obscured or partially obscured cells
IL85532A (en) * 1987-03-13 1992-03-29 Coulter Electronics Method and lytic reagent system for isolation,identification and/or analysis of leukocytes from whole blood samples
US5389549A (en) * 1987-05-29 1995-02-14 Toa Medical Electronics Co., Ltd. Method for classifying leukocytes and a reagent used therefor
JP2635142B2 (en) * 1987-05-29 1997-07-30 東亜医用電子株式会社 Leukocyte classification method and reagent
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US5408307A (en) * 1988-07-11 1995-04-18 Omron Tateisi Electronics Co. Cell analyzer
US5089384A (en) * 1988-11-04 1992-02-18 Amoco Corporation Method and apparatus for selective cell destruction using amplified immunofluorescence
CA2016699C (en) * 1989-05-15 2003-11-18 Paul N. Marshall Lytic agents and uses thereof
JP2815435B2 (en) * 1989-12-22 1998-10-27 株式会社日立製作所 Particle analyzer and blood cell counter
JP2941041B2 (en) * 1990-11-16 1999-08-25 シスメックス株式会社 Classification of leukocytes by flow cytometry
US5270208A (en) * 1991-05-09 1993-12-14 Streck Laboratories, Inc. White blood cell hematology control
US5316725A (en) * 1991-06-07 1994-05-31 Edward Lawrence Carver, Jr. Reagent system for the improved determination of white blood cell subpopulations
US5262329A (en) * 1991-06-13 1993-11-16 Carver Jr Edward L Method for improved multiple species blood analysis
JP3048260B2 (en) * 1991-07-29 2000-06-05 シスメックス株式会社 Sample preparation method for leukocyte classification and counting
JP3067849B2 (en) * 1991-07-29 2000-07-24 シスメックス株式会社 Sample preparation method for leukocyte classification and counting
US5776709A (en) * 1991-08-28 1998-07-07 Becton Dickinson And Company Method for preparation and analysis of leukocytes in whole blood
WO1993016384A1 (en) * 1992-02-07 1993-08-19 Abbott Laboratories Method for accurately enumerating and sensitively qualifying heterogeneous cell populations in cytolytic processing conditions
US5451525A (en) * 1992-02-14 1995-09-19 Coulter Corporation Method and materials for determining particle count in a flow cytometer
JP3391451B2 (en) * 1992-02-24 2003-03-31 クールター インターナショナル コーポレイション Blood control composition for leukocyte analogs, and method of preparation and use thereof
ATE193603T1 (en) * 1992-02-24 2000-06-15 Coulter Corp SUSPENSION MEDIA FOR HEMATOLIGIC COMPOSITIONS AND METHOD FOR THEIR USE
US6509192B1 (en) * 1992-02-24 2003-01-21 Coulter International Corp. Quality control method
US6362003B1 (en) 1992-02-24 2002-03-26 Coulter Corporation Hematological reference control composition containing leukocyte analogs, methods of making, and uses thereof
DE69434942T2 (en) * 1993-02-25 2007-11-29 Abbott Laboratories, Abbott Park MULTIPURPOSE REAGENT SYSTEM FOR THE FAST LYSING OF FULL BLOOD SAMPLES
JP3301646B2 (en) 1993-03-19 2002-07-15 シスメックス株式会社 Reagent for immature cell measurement
ATE236397T1 (en) * 1993-05-14 2003-04-15 Coulter Int Corp RETICULOCYTE DETERMINATION METHOD AND DEVICE USING LIGHT SCATTERING TECHNIQUES
US5631165A (en) * 1994-08-01 1997-05-20 Abbott Laboratories Method for performing automated hematology and cytometry analysis
US5656499A (en) * 1994-08-01 1997-08-12 Abbott Laboratories Method for performing automated hematology and cytometry analysis
US5891734A (en) * 1994-08-01 1999-04-06 Abbott Laboratories Method for performing automated analysis
US5631730A (en) * 1994-08-01 1997-05-20 Abbott Laboratories Pseudo telecentric optical design for flow cytometric blood cell analyzer
US5879900A (en) * 1994-12-15 1999-03-09 Abbott Laboratories Method for simultaneous analysis of cell viability, nucleated red blood cells and white blood cell differentials
US5559037A (en) * 1994-12-15 1996-09-24 Abbott Laboratories Method for rapid and simultaneous analysis of nucleated red blood cells
FR2735578B1 (en) * 1995-06-13 1997-09-05 Hycel Groupe Lisabio ERYTHROCYTE LYSIS REAGENT, AND USE THEREOF IN LEUKOCYTE ISOLATION AND DISCRIMINATION PROCESSES
US5817518A (en) * 1995-12-18 1998-10-06 Coulter International Corp. Reagent and method for differential determination of leukocytes in blood
US6146901A (en) * 1997-06-16 2000-11-14 Hematronix, Inc. Composition for manipulating optical and electrical properties of particles to achieve target values for such properties and methods for using the composition
US6106779A (en) * 1997-10-02 2000-08-22 Biosite Diagnostics, Inc. Lysis chamber for use in an assay device
FR2781055B1 (en) * 1998-07-09 2000-10-13 Immunotech Sa REAGENT AND METHOD FOR PERMEABILIZATION AND IDENTIFICATION OF ERYTHROCYTES
FR2782166B1 (en) * 1998-08-04 2000-10-06 Abx Sa REAGENT FOR MEASURING HEMOGLOBIN AND DETERMINING LEUKOCYTES IN A BLOOD SAMPLE
FR2782730B1 (en) * 1998-08-25 2002-05-17 Biocom Sa CELL SEPARATION PROCESS FOR THE ISOLATION OF PATHOGENIC CELLS, PARTICULARLY RARE CANCERES, EQUIPMENT AND REAGENT FOR IMPLEMENTING THE PROCESS AND APPLICATION OF THE PROCESS
US6221668B1 (en) 1999-08-20 2001-04-24 Streck Laboratories, Inc. Hematology control and system for multi-parameter hematology measurements
US6200500B1 (en) 1999-08-20 2001-03-13 Streck Laboratories, Inc. Hematology control and system for multi-parameter hematology measurements
US20020098589A1 (en) * 2001-01-19 2002-07-25 Crews Harold Richardson Multi-purpose reagent system and method for enumeration of red blood cells, white blood cells and thrombocytes and differential determination of white blood cells
US6514763B2 (en) * 2000-04-28 2003-02-04 Hematronix, Inc. Hematology blood control and method for preparation of same
WO2002063367A1 (en) * 2001-02-02 2002-08-15 Cellomics, Inc. Method for estimating the best initial focus position
FR2821428B1 (en) * 2001-02-23 2004-08-06 Abx Sa REAGENT AND METHOD FOR THE IDENTIFICATION AND COUNTING OF BIOLOGICAL CELLS
US20030119080A1 (en) * 2001-10-15 2003-06-26 Mangano Joseph A. Strategies for the identification and isolation of cancer stem cells and non-cancerous stem cells
US6653137B2 (en) 2001-12-03 2003-11-25 Streck Laboratories Inc. Hematology reference control
US6723563B2 (en) 2001-12-03 2004-04-20 Streck Laboratories Inc. Hematology reference control
US7771658B2 (en) * 2002-06-11 2010-08-10 Chempaq A/S Disposable cartridge for characterizing particles suspended in a liquid
US9133024B2 (en) * 2003-09-03 2015-09-15 Brigitte Chau Phan Personal diagnostic devices including related methods and systems
CA2597496A1 (en) 2005-02-10 2006-08-17 Chempaq A/S Dual sample cartridge and method for characterizing particles in liquid
CA2855108A1 (en) * 2005-02-10 2006-08-17 Koninklijke Philips Electronics N.V. Dual sample cartridge and method for characterizing particles in liquid
WO2007140963A1 (en) 2006-06-06 2007-12-13 Roche Diagnostics Gmbh Ready-to-use whole blood collection vessel
CA2649633C (en) 2006-06-06 2012-11-27 F. Hoffmann-La Roche Ag Differential hemolysis of a whole blood sample
WO2008030154A1 (en) * 2006-09-08 2008-03-13 Hemocue Ab Haemolysing agent
CN101349644B (en) * 2007-07-20 2012-06-27 深圳迈瑞生物医疗电子股份有限公司 Leukocytes classification agent and use method thereof
CN101464454B (en) * 2007-12-18 2016-08-03 深圳迈瑞生物医疗电子股份有限公司 Leucocyte classification reagent and method
CN105440725A (en) * 2008-01-04 2016-03-30 深圳迈瑞生物医疗电子股份有限公司 Asymmetric cyanine fluorescent dye, composition and application in biological sample dyeing
CN101602762B (en) * 2008-06-10 2013-10-16 深圳迈瑞生物医疗电子股份有限公司 Asymmetric cyanine compound, preparation method and application thereof
US20100086962A1 (en) * 2008-10-08 2010-04-08 Streck, Inc. Hematology controls and methods
CN101726579B (en) * 2008-10-17 2014-06-18 深圳迈瑞生物医疗电子股份有限公司 Blood test reagent and method
US8367358B2 (en) * 2008-12-17 2013-02-05 Shenzhen Mindray Bio-Medical Electronics Co., Ltd. Reagent, kit and method for differentiating and counting leukocytes
EP2259044A1 (en) * 2009-06-05 2010-12-08 Koninklijke Philips Electronics N.V. Multi-frequency impedance method and apparatus for discriminating and counting particles expressing a specific marker
EP2259045A1 (en) * 2009-06-05 2010-12-08 Koninklijke Philips Electronics N.V. Multi-frequency impedance method and apparatus for discriminating and counting particles expressing a specific marker
CN101988082B (en) * 2009-07-31 2015-04-08 深圳迈瑞生物医疗电子股份有限公司 Leukocyte classified counting reagent, kit and preparation method thereof and method of leukocyte classified counting
JP6692743B2 (en) * 2013-03-12 2020-05-13 アボット・ラボラトリーズAbbott Laboratories Reagents, systems, and methods for analyzing white blood cells
CN106687810B (en) * 2014-12-31 2019-10-22 深圳迈瑞生物医疗电子股份有限公司 A kind of erythroblast alarm method, device and the stream type cell analyzer of non-diagnostic purpose
JP7143678B2 (en) * 2018-08-24 2022-09-29 東ソー株式会社 Methods for detecting target cells
CN116097093A (en) * 2020-09-03 2023-05-09 生物辐射实验室股份有限公司 Preparation of Nucleated RBC (NRBC) analogs for use as reference blood controls in automated blood analyzers

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3502974A (en) * 1966-05-23 1970-03-24 Coulter Electronics Signal modulated apparatus for generating and detecting resistive and reactive changes in a modulated current path for particle classification and analysis
US3710933A (en) * 1971-12-23 1973-01-16 Atomic Energy Commission Multisensor particle sorter
US3884579A (en) * 1973-06-14 1975-05-20 Cambridge Chemical Products In Method for counting blood platelets
US4146604A (en) * 1973-05-31 1979-03-27 Block Engineering, Inc. Differential counting of leukocytes and other cells
US4286963A (en) * 1979-11-23 1981-09-01 Coulter Electronics, Inc. Differential lymphoid-myeloid determination of leukocytes in whole blood

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2656508A (en) * 1949-08-27 1953-10-20 Wallace H Coulter Means for counting particles suspended in a fluid
US3259842A (en) * 1959-08-19 1966-07-05 Coulter Electronics Particle analyzing device
US3446751A (en) * 1965-11-29 1969-05-27 Brunswick Corp Hemolyzing composition
US3741875A (en) * 1970-10-30 1973-06-26 Mount Sinai Res Foundation Inc Process and apparatus for obtaining a differential white blood cell count
BE790042A (en) * 1971-10-14 1973-04-13 Coulter Electronics METHOD FOR CLASSIFYING PARTICLES
US3883247A (en) * 1973-10-30 1975-05-13 Bio Physics Systems Inc Method for fluorescence analysis of white blood cells
US3873467A (en) * 1974-02-01 1975-03-25 United Medical Lab Inc Hematologic reference control
US4198206A (en) * 1977-06-13 1980-04-15 Ryan Wayne L Method for preparing a platelet reference control
US4099917A (en) * 1977-07-21 1978-07-11 Technicon Instruments Corporation Process for preparing a cell suspension from blood for discrimination of white blood cells and platelets from other blood particles
US4213876A (en) * 1978-08-22 1980-07-22 Coulter Electronics, Inc. Multi-purpose blood diluent for use in electronic blood analysis instrumentation
US4250051A (en) * 1978-12-26 1981-02-10 Coulter Electronics, Inc. Preservative for use in calibrator compositions for blood analysis
US4521518A (en) * 1980-06-16 1985-06-04 Coulter Electronics, Inc. Multi-purpose blood diluent and lysing agent for differential determination of lymphoid-myeloid population of leukocytes
US4500509A (en) * 1981-03-11 1985-02-19 Lawrence Kass Metachromatic dye sorption and fluorescent light emmisive means for differential determination of developmental stages of neutrophilic granulocytic cells and other leukocytes
US4492752A (en) * 1982-09-03 1985-01-08 Ortho Diagnostics Systems Inc. Method for discriminating between unstained and absorbing dye stained cells
US4506018A (en) * 1982-12-30 1985-03-19 Becton, Dickinson And Company Blood diluent
US4485175A (en) * 1983-01-03 1984-11-27 Coulter Electronics, Inc. Method for three-volume differential determination of lymphocyte, monocyte and granulocyte populations of leukocytes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3502974A (en) * 1966-05-23 1970-03-24 Coulter Electronics Signal modulated apparatus for generating and detecting resistive and reactive changes in a modulated current path for particle classification and analysis
US3710933A (en) * 1971-12-23 1973-01-16 Atomic Energy Commission Multisensor particle sorter
US4146604A (en) * 1973-05-31 1979-03-27 Block Engineering, Inc. Differential counting of leukocytes and other cells
US3884579A (en) * 1973-06-14 1975-05-20 Cambridge Chemical Products In Method for counting blood platelets
US4286963A (en) * 1979-11-23 1981-09-01 Coulter Electronics, Inc. Differential lymphoid-myeloid determination of leukocytes in whole blood

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Clinical Chemistry, Volume 23, issued 1977, LEIF et al, Development of Instrumentation and Fluorochromes for Automated Multiparameter Analysis of Cells, pages 1492-1498 *
Ser. Haemat., Volume 2, issued 1972, HUMPHRIES et al, Volume Analysis of Human Peripheral Blood Leukocytes, pages 142-162 *
The Condensed Chemical Dictionary, Tenth Edition, issued 1981, Revised by G. HAWLEY, New York, Van NOSTRAND REINHOLD Company, page 959 *
The Journal of Histochemistry and Cytochemistry, Volume 25, Number 7, THOMAS et al, Combined Optical and Electronic Analysis of Cells with the AMAC Transducers, pages 827-835 *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0259834A2 (en) * 1986-09-10 1988-03-16 Toa Medical Electronics Co., Ltd. Method of classifying leucocytes by flow cytometry
EP0259834A3 (en) * 1986-09-10 1988-12-07 Toa Medical Electronics Co., Ltd. Method of classifying leucocytes by flow cytometry
US4933293A (en) * 1986-09-10 1990-06-12 Toa Medical Electronics Co., Ltd. Method of classifying leukocytes by flow cytometry and reagents used in the method
US4791355A (en) * 1986-10-21 1988-12-13 Coulter Electronics Inc. Particle analyzer for measuring the resistance and reactance of a particle
EP0483116A2 (en) * 1986-11-27 1992-04-29 Toa Medical Electronics Co., Ltd. Method of classifying leukocytes by flow cytometry and reagents used in the method
EP0483116A3 (en) * 1986-11-27 1992-07-22 Toa Medical Electronics Co., Ltd. Method of classifying leukocytes by flow cytometry and reagents used in the method
WO1988007198A1 (en) * 1987-03-13 1988-09-22 Coulter Electronics, Inc. Multi-part differential analyzing apparatus utilizing light scatter techniques
FR2654744A1 (en) * 1989-11-20 1991-05-24 Abx Sa REAGENT AND METHOD OF USE THEREOF FOR THE AUTOMATIC DETERMINATION IN CYTOMETRY OF FLOWS OF AT LEAST ONE LEUKOCYTE SUB-POPULATION FROM TOTAL BLOOD.
EP0430750A1 (en) * 1989-11-20 1991-06-05 ABX , Société Anonyme dite Reagent and its use in flow cytometry for automatically determining at least one leukocyte subpopulation obtained from whole blood
US5232857A (en) * 1989-11-20 1993-08-03 Abx Reagent for use in automatic analyzers for distinguisher leukocyte sub-populations in blood samples
EP0574267A2 (en) * 1992-06-12 1993-12-15 Gen-Probe Incorporated Preparation of nucleic acid from blood
EP0574267A3 (en) * 1992-06-12 1994-08-24 Gen Probe Inc Preparation of nucleic acid from blood
EP0582736A1 (en) * 1992-08-11 1994-02-16 Abbott Laboratories Flow cytometry sheath reagent for elimination of red cells with preservation of nucleated cells
CN1040469C (en) * 1992-11-19 1998-10-28 东亚医用电子株式会社 A pretreatment method for blood analysis
EP0642022A2 (en) * 1993-09-07 1995-03-08 Heinrich Prof. Dr. Patscheke Stabilizing medium for blood
EP0642022A3 (en) * 1993-09-07 1995-11-29 Patscheke Heinrich Stabilizing medium for blood.
WO1996034291A1 (en) * 1995-04-28 1996-10-31 Coulter International Corp. Quality control method of hematology instruments
DE19520298A1 (en) * 1995-06-02 1996-12-05 Bayer Ag Sorting device for biological cells or viruses
US5837200A (en) * 1995-06-02 1998-11-17 Bayer Aktiengesellschaft Sorting device for biological cells or viruses
WO1998026284A1 (en) * 1996-12-11 1998-06-18 Nycomed Amersham Plc Selective lysis of cells
EP2278331A1 (en) * 2008-05-02 2011-01-26 ARKRAY, Inc. Leukocyte analysis method and analysis reagent for use in the method
EP2278331A4 (en) * 2008-05-02 2013-03-13 Arkray Inc Leukocyte analysis method and analysis reagent for use in the method
EP3719141A1 (en) * 2015-04-20 2020-10-07 QIAGEN GmbH Method, lysis solution and kit for selectively depleting animal nucleic acids in a sample

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US4751179A (en) 1988-06-14
ES543657A0 (en) 1986-08-16
AU4401985A (en) 1985-12-31
CA1248856A (en) 1989-01-17
DE3587036T2 (en) 1993-09-02
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ES8609727A1 (en) 1986-08-16
JPS61502277A (en) 1986-10-09

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