CA1248856A - Method and reagent system for four-population differential determination of leukocytes - Google Patents
Method and reagent system for four-population differential determination of leukocytesInfo
- Publication number
- CA1248856A CA1248856A CA000482841A CA482841A CA1248856A CA 1248856 A CA1248856 A CA 1248856A CA 000482841 A CA000482841 A CA 000482841A CA 482841 A CA482841 A CA 482841A CA 1248856 A CA1248856 A CA 1248856A
- Authority
- CA
- Canada
- Prior art keywords
- reagent
- saponin
- volume
- reagent system
- lytic
- Prior art date
- Legal status (The legal status 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 status listed.)
- Expired
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5094—Chemical 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
- Y10T436/101666—Particle count or volume standard or control [e.g., platelet count standards, etc.]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
- Y10T436/107497—Preparation composition [e.g., lysing or precipitation, etc.]
Abstract
ABSTRACT
This invention describes 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.
This invention describes 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
.5~
Many methods have been used for removin~ 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 purposeg, 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.
Rim, 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 58C, to lysP 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, wfiich require more thorough 3tromatolyzing of the red blood cells. The same is true of other lytic procedures, such as 2Q hypotonic lysis, ammonium chloride lysis, and ethylene or propylene glycol treatment which render the red blood cells transparent towards optical, i.e. ligh~ scatter, fluorescence measurements, but not towards electronic, i.e. DC volume and R.F. volume parameters.
The natural product known as sapoain has long been used as a red blood cell lytic agent. Saponin is chemically deined 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. Thi3 saponin ha~ detergent-like properties and hemoly~es 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 te~ed, and the material does vary from lot to lot.
The activity of saponin i9 more selective toward~ red blood cells than are the quaternary a~monium salt detergents. Unfortunately, by .. . .
, .
employing lysing procedureg known heretofore, it has not been possible to obtain leukocyte6 free from red blood cells, without doing some concomitant damage to the leukocytes.
Several reports describe the use of saponin with a second reagent ~hich retards the leukocyte damage. Hughes-Jone~, J. Clin. Path., Vol. 27, pa~e 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, pa~e 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 ~ith 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
Many methods have been used for removin~ 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 purposeg, 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.
Rim, 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 58C, to lysP 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, wfiich require more thorough 3tromatolyzing of the red blood cells. The same is true of other lytic procedures, such as 2Q hypotonic lysis, ammonium chloride lysis, and ethylene or propylene glycol treatment which render the red blood cells transparent towards optical, i.e. ligh~ scatter, fluorescence measurements, but not towards electronic, i.e. DC volume and R.F. volume parameters.
The natural product known as sapoain has long been used as a red blood cell lytic agent. Saponin is chemically deined 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. Thi3 saponin ha~ detergent-like properties and hemoly~es 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 te~ed, and the material does vary from lot to lot.
The activity of saponin i9 more selective toward~ red blood cells than are the quaternary a~monium salt detergents. Unfortunately, by .. . .
, .
employing lysing procedureg known heretofore, it has not been possible to obtain leukocyte6 free from red blood cells, without doing some concomitant damage to the leukocytes.
Several reports describe the use of saponin with a second reagent ~hich retards the leukocyte damage. Hughes-Jone~, J. Clin. Path., Vol. 27, pa~e 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, pa~e 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 ~ith 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~J and the principle of their operation is co~monly known as the Coulter principle.
According to the Coulter principle, first patented in U. S.
Patent 2,656,50a, 1953, when a particle o 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 ~ If the electrical field is excited by a direct (DC) or low frequency current, the electrical change i8 closely proportional to the volume of the particle. In commerical~apparatus, the changes are detected by some suitable means and used to operate couneers and analyzers. The analy~ers associated with such~ spparatus classify and 3ize psrticles 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, u~ing radio frequency ~RF) current in addition to DC current field excitation to~provide not only DC volume information ::
~ :, .
concerning the particle studied, but al80 information due to the composition and na~ure of the material constituting the particle.
This patent discloses apparatus capable of distinguishing between particles of identical size, but of different material. By generating ! 5 the particle sensing Eield by means of both a lo~ 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 impe~ing 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 ~C current goes around a blood cell, some of the RF current ~ill 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 t impede RF current is termed its "opaci~y". In later publications, ; ~ "opacity" iB defined as the RF - ~ divided by the DC ~ ~ ~e The relative electrical opacity of a particle becomes an identifying feature of the particle contents, and hence, its particle type for classification purpose~. To the extent that different types of particles each possess a different opacity, the difference between them is detectable. Howeverj significsntly different particles can possess substantially the same opacity and such particles cannot be classified effectivaly in this manner. In ~. S.~Patent 3,836,849, 1974, Coulter et al t~ught that it is possible to cbange selectively the opacity of particle types by treatment of the particles, 80 that detectable differences result.
Although red blood cells and white blood cells nominally have different size}l their slze ranges teod to overlap, or at le~}t under .
, .5~
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 ~tep 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 speci~ic substrates for diferent enzymes contained in specific kinds of cells, absolute and relative counts of the different kinds of cells are obtained. The cytological fixing ~olution 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 hemoly~e the red blood cells, since there i~ danger that coincident pa~sage of two or more red cell~ 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 o~ a reagent to the suspen~ion of cells to cause the red blood cell~ to rupture and release their hemoglobin content into the solution.
Ledi~ et al, U. SO Patent 4,286,963, 1981, teaches a method for two-volume snalysis of leukocytes using a COULTER COUNTER analyzer which employ~ 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 rea8ent 8y8tem for three-volume differential determination of lymphocyte, =onocyte, and granulocy~e populations of leukocytes, u~ing quaternary ammonium ~alt~ as lysing agent~ and the COULTER COUNTER Model S Plus automated blood counter, which instrument employs only direct current field excitation.
, ~ ;3 ~
Previous methods of flow analysi~ of leukocytes using DC volume, or light scatter at various angles have shown only three clusters of l~ukocytes, 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 compcsitions 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
COUNTER~J and the principle of their operation is co~monly known as the Coulter principle.
According to the Coulter principle, first patented in U. S.
Patent 2,656,50a, 1953, when a particle o 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 ~ If the electrical field is excited by a direct (DC) or low frequency current, the electrical change i8 closely proportional to the volume of the particle. In commerical~apparatus, the changes are detected by some suitable means and used to operate couneers and analyzers. The analy~ers associated with such~ spparatus classify and 3ize psrticles 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, u~ing radio frequency ~RF) current in addition to DC current field excitation to~provide not only DC volume information ::
~ :, .
concerning the particle studied, but al80 information due to the composition and na~ure of the material constituting the particle.
This patent discloses apparatus capable of distinguishing between particles of identical size, but of different material. By generating ! 5 the particle sensing Eield by means of both a lo~ 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 impe~ing 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 ~C current goes around a blood cell, some of the RF current ~ill 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 t impede RF current is termed its "opaci~y". In later publications, ; ~ "opacity" iB defined as the RF - ~ divided by the DC ~ ~ ~e The relative electrical opacity of a particle becomes an identifying feature of the particle contents, and hence, its particle type for classification purpose~. To the extent that different types of particles each possess a different opacity, the difference between them is detectable. Howeverj significsntly different particles can possess substantially the same opacity and such particles cannot be classified effectivaly in this manner. In ~. S.~Patent 3,836,849, 1974, Coulter et al t~ught that it is possible to cbange selectively the opacity of particle types by treatment of the particles, 80 that detectable differences result.
Although red blood cells and white blood cells nominally have different size}l their slze ranges teod to overlap, or at le~}t under .
, .5~
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 ~tep 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 speci~ic substrates for diferent enzymes contained in specific kinds of cells, absolute and relative counts of the different kinds of cells are obtained. The cytological fixing ~olution 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 hemoly~e the red blood cells, since there i~ danger that coincident pa~sage of two or more red cell~ 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 o~ a reagent to the suspen~ion of cells to cause the red blood cell~ to rupture and release their hemoglobin content into the solution.
Ledi~ et al, U. SO Patent 4,286,963, 1981, teaches a method for two-volume snalysis of leukocytes using a COULTER COUNTER analyzer which employ~ 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 rea8ent 8y8tem for three-volume differential determination of lymphocyte, =onocyte, and granulocy~e populations of leukocytes, u~ing quaternary ammonium ~alt~ as lysing agent~ and the COULTER COUNTER Model S Plus automated blood counter, which instrument employs only direct current field excitation.
, ~ ;3 ~
Previous methods of flow analysi~ of leukocytes using DC volume, or light scatter at various angles have shown only three clusters of l~ukocytes, 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 compcsitions 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 suspension9 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 partirles 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 a-nd their alkyl, alkoxy or halogen substituted derivatives.
~0 The development of instrumentation and fluorochromes for autom~ted multiparameter analysis of cells is further described by R.C. Leif et al. in Clinical Chemi3try, Vol. 23, pp 1492-98 (1977).
Eosinophils have been observed also by en~yme stsining such as by the Technicon Hemalog D and H6000 instruments, (An31ey and Ornstein, Adv. Automflted Anal., Vol. 1 437 S1971), 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 disesses. Such data are useful as a ~creening tool for calling attention to abnormal leukocyte ratios. Abnor~al situations identified by this method give information of diagnostio significance and alert the technologise to the need for further study.
This invention concerns a reagent system and a method for cla~sifying ~nd counting at lea~t four populations of leukocytes usine 5~i flow analysis instrumentation. More particularly, the present invention relates to a multicomponent reagent ~ystem 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 ~ollowed by a lytic reagent containing saponin; (B) a fixing reagent containing a cross-linking compound such as glutaraldehyde, 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.
Preservative3 can be added to inhibit the growth of microorganisms. Other additives al~o can be included. A fluorescent dye is included in the lytic diluent when ~tudies make a determination by fluorescence.-The lysed and fixed blood sample i9 brought to the proper ~0 concentration for flow analysi~ using a diluent which also can contain potassium ferricyanide and potas8ium cyanide for converting the hemo~lobin to a ~ suitable for hemoglobinometry.
This invention relates also to a method for discriminating of major categories of leukocytes by flow in~trumentation which measures two or more of the primary parameter8 of DC volume, RF ~ize, fluorescence and light 8catter, and also provides hi8tograms bas2d on selected mathematical combinations of the primary parameters.
By way of example, illu8trative embodiment~ of the invention now will be described with reference to the accomp~nying drawings in which: ~
FIGURES 1 through 5 illu3trate the leukocyte subpopulation identification accomplished by the method of this invention. In the dia8ram8 of the FIGURES~ the encircled area~ repre~ent cell `` populations or cell clusters that ara generated by accu~ulation of data point8, in which each da~a point i8 determined by coordlnates f~ f~
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 modiies 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 ewo forms: (1) a lytic diluent containing saponin, which simultaneously functions to dilute the whole blood sample and 3tromatolyse its red blood cells; or (2) a two part system co~prised of non-lytic blood diluent followed by a lytic reagent containing saponin. The lysing reagent i8 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 excita~ion. The desired parameters can be measured directly, or by a mathematical calculation from the direct measurements.
FIGURÆS 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 do~ repre~ents a single cell, and the location of the dot is given by coortinates which are proportional to selected cell par~meters; for example, the right angle and forward light scatter intensities produced by the cell in the instrum&nt. In thi~
manner, four clusters of dots or cells are formed, and eheir areas are encircled in FIGURES 1 through 5 and all of which are identiflsd as leukocytes, namely (1) lymphocyte~, (2) monocytes, (3) neu~rophils and
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 partirles 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 a-nd their alkyl, alkoxy or halogen substituted derivatives.
~0 The development of instrumentation and fluorochromes for autom~ted multiparameter analysis of cells is further described by R.C. Leif et al. in Clinical Chemi3try, Vol. 23, pp 1492-98 (1977).
Eosinophils have been observed also by en~yme stsining such as by the Technicon Hemalog D and H6000 instruments, (An31ey and Ornstein, Adv. Automflted Anal., Vol. 1 437 S1971), 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 disesses. Such data are useful as a ~creening tool for calling attention to abnormal leukocyte ratios. Abnor~al situations identified by this method give information of diagnostio significance and alert the technologise to the need for further study.
This invention concerns a reagent system and a method for cla~sifying ~nd counting at lea~t four populations of leukocytes usine 5~i flow analysis instrumentation. More particularly, the present invention relates to a multicomponent reagent ~ystem 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 ~ollowed by a lytic reagent containing saponin; (B) a fixing reagent containing a cross-linking compound such as glutaraldehyde, 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.
Preservative3 can be added to inhibit the growth of microorganisms. Other additives al~o can be included. A fluorescent dye is included in the lytic diluent when ~tudies make a determination by fluorescence.-The lysed and fixed blood sample i9 brought to the proper ~0 concentration for flow analysi~ using a diluent which also can contain potassium ferricyanide and potas8ium cyanide for converting the hemo~lobin to a ~ suitable for hemoglobinometry.
This invention relates also to a method for discriminating of major categories of leukocytes by flow in~trumentation which measures two or more of the primary parameter8 of DC volume, RF ~ize, fluorescence and light 8catter, and also provides hi8tograms bas2d on selected mathematical combinations of the primary parameters.
By way of example, illu8trative embodiment~ of the invention now will be described with reference to the accomp~nying drawings in which: ~
FIGURES 1 through 5 illu3trate the leukocyte subpopulation identification accomplished by the method of this invention. In the dia8ram8 of the FIGURES~ the encircled area~ repre~ent cell `` populations or cell clusters that ara generated by accu~ulation of data point8, in which each da~a point i8 determined by coordlnates f~ f~
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 modiies 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 ewo forms: (1) a lytic diluent containing saponin, which simultaneously functions to dilute the whole blood sample and 3tromatolyse its red blood cells; or (2) a two part system co~prised of non-lytic blood diluent followed by a lytic reagent containing saponin. The lysing reagent i8 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 excita~ion. The desired parameters can be measured directly, or by a mathematical calculation from the direct measurements.
FIGURÆS 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 do~ repre~ents a single cell, and the location of the dot is given by coortinates which are proportional to selected cell par~meters; for example, the right angle and forward light scatter intensities produced by the cell in the instrum&nt. In thi~
manner, four clusters of dots or cells are formed, and eheir areas are encircled in FIGURES 1 through 5 and all of which are identiflsd as leukocytes, namely (1) lymphocyte~, (2) monocytes, (3) neu~rophils and
(4) eosinophils, are the four major cstegories of leukoc~tes.
Because a hi8h concentration o~ saponin is needed to provide `` rapid stromatolyzing of the red cell~, ie mu~t be used with a rapidly active cross-linking fixing reagent in order to proteet the leukocyteo ' , 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 a~ter stromatolysing the red blood cells.
In the examples which follow, the formulations can be adjusted to -~ake into account certain general considerations.
Since commercial saponin is not a pure ma~erial, 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, i~ a compensating change in volume of the saponin reagent is also made. The ~aponin concentration in ths lytic diluent ordinarily is within the range of ~.15% to 0.40% wtv.
When the blood sample is prediluted with a non-lytic diluent, t~e lytic reagent can contain a higher concentration of saponin, approximately 0.30% to 4.0%, and the alkaline buffer i8 best included in the prediluent.
Preservatives can be added to the lytic diluent and the lytic reagent to inhibit the growth of mic~roorganisms. ~ater soluble preservatives, such as methyl paraben, propyl paraben, formaldehyde, acetaldehydel dimethylolurea, 2-pyridinethiol-1-oxide, sorbic scid, and potassium sorbate can be used.
Additives to the lysing reagent substantially can enhance the separation of the four main white blood cell cluster~. 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 1-phenyl-2-propanol, 2-phenyl-1-propanol, 3-phenoxy-1-propanol and 3-phenyl-1-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 a8 glucose, `~ lactose, and ~ucrose, in the concentration range of 2 to 8X. Mixtures of more than one additive ~ill give ~t ~i~es i~proved results.
, ' ~
The saponin lytic diluent i9 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 fluorei3cent dye is included as is discussed below.
Generally, cyanine dyes are employed which contsin sulfonic acid side chains and which suitably fluoresce by excitation in the-blue to green range 488 eo 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 10-6 M to 5 x 10-5 M:
Di I C3S03-(3) Di O C3S03-(3) Di S C3S03-(3) OC S03 = TBA-Cl (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 ~ 10 5 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, sulforhodamine B.
The abbreviation~ for the dyes are those ui3ed by Alan ~aggoner, Biochem 13 3315 (1974). He/Ar means Helium/Argon; whereas, ~e/Ne means Helium/Neon.
Glutaraldehyde is the preferred cross-linking fi~ing reagent.
Other crosæ-linking dialdehydes include glyoxal, malonaldehyde, and the like. Unsaturated monoaldehydes, such as acrolein or methacrolein, likewise can be used. I~ is known that acrolein under certain conditionc iæ a bifunctional cro~s-linking agent.
The preferred cross-linking reagent which is added to the lyàed blood s$mple contain~ glutaraldehyde in the concentr~tion range of 0.5 to 4.0% v/v. This solution also contains ~odium chloride in the range of 0.2 to 0.6~ w/v and a buffer is included to maint~in the pH
~, .
Because a hi8h concentration o~ saponin is needed to provide `` rapid stromatolyzing of the red cell~, ie mu~t be used with a rapidly active cross-linking fixing reagent in order to proteet the leukocyteo ' , 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 a~ter stromatolysing the red blood cells.
In the examples which follow, the formulations can be adjusted to -~ake into account certain general considerations.
Since commercial saponin is not a pure ma~erial, 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, i~ a compensating change in volume of the saponin reagent is also made. The ~aponin concentration in ths lytic diluent ordinarily is within the range of ~.15% to 0.40% wtv.
When the blood sample is prediluted with a non-lytic diluent, t~e lytic reagent can contain a higher concentration of saponin, approximately 0.30% to 4.0%, and the alkaline buffer i8 best included in the prediluent.
Preservatives can be added to the lytic diluent and the lytic reagent to inhibit the growth of mic~roorganisms. ~ater soluble preservatives, such as methyl paraben, propyl paraben, formaldehyde, acetaldehydel dimethylolurea, 2-pyridinethiol-1-oxide, sorbic scid, and potassium sorbate can be used.
Additives to the lysing reagent substantially can enhance the separation of the four main white blood cell cluster~. 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 1-phenyl-2-propanol, 2-phenyl-1-propanol, 3-phenoxy-1-propanol and 3-phenyl-1-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 a8 glucose, `~ lactose, and ~ucrose, in the concentration range of 2 to 8X. Mixtures of more than one additive ~ill give ~t ~i~es i~proved results.
, ' ~
The saponin lytic diluent i9 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 fluorei3cent dye is included as is discussed below.
Generally, cyanine dyes are employed which contsin sulfonic acid side chains and which suitably fluoresce by excitation in the-blue to green range 488 eo 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 10-6 M to 5 x 10-5 M:
Di I C3S03-(3) Di O C3S03-(3) Di S C3S03-(3) OC S03 = TBA-Cl (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 ~ 10 5 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, sulforhodamine B.
The abbreviation~ for the dyes are those ui3ed by Alan ~aggoner, Biochem 13 3315 (1974). He/Ar means Helium/Argon; whereas, ~e/Ne means Helium/Neon.
Glutaraldehyde is the preferred cross-linking fi~ing reagent.
Other crosæ-linking dialdehydes include glyoxal, malonaldehyde, and the like. Unsaturated monoaldehydes, such as acrolein or methacrolein, likewise can be used. I~ is known that acrolein under certain conditionc iæ a bifunctional cro~s-linking agent.
The preferred cross-linking reagent which is added to the lyàed blood s$mple contain~ glutaraldehyde in the concentr~tion range of 0.5 to 4.0% v/v. This solution also contains ~odium chloride in the range of 0.2 to 0.6~ w/v and a buffer is included to maint~in the pH
~, .
5~
between 7.0 and 8Ø 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/~.
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 solu~ion i8 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-4~*ge~, 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 75C for about 10 second~ 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 75C water bath wieh swirling to achieve heatin8.
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.
FO~MULATIONS
Lytic diluent 0.2~ w/v saponin 0.6% w/v sodium chloride l.OX ~epe~ Buffer Cyanine dye 1 ~ 10 5 ~ Di l-C3S03-(3) adjust pH to 7.2 wieh sodium hydroxide.
~ Fixing reagent 0.5% v/v glutaraldehyde 1,5: v/v so~ bicsrbona-e 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 ~en seconds, 2.0 ml of the fixing r~agent is added with swirling. After fifteen seconds the mixture is heated in a 70~C water bath for fifteen seconds with swirling.
The preparation i8 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 ~ass filter are placed in the fluorescence channel~ The white blood cell histogram i3 collected as fluorescence vs light scatter (2 to 20), as shown in FIGU~E 1, or as fluorescence/light scaeter V8 light scatter.
Four distinct clusters of white blood cells are observed.
Sorting of cells in each of these clus~ers and microscopic examination shows that the cells are lymphocytes at-low 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 i8 observed at low light scatter-intermediate fluorescence.
Example 2 White blood cell histogram by fluorescence and light scatter using a COULTER TPS-l equipped with 30 mwatt HeNe laser at 633 nm.
FORMULATIO~S
Lytic diluent 0.2% w/v saponin 0.5% w/v sodium ~ulfate 3.0% w/v sucrose cyanine dye 1 x 10-5 M DiIC3S03-(5) Fixîng reagent 1.0% v/v glutsraldehyde 0.8% w/v sodium chloride 0.5% w/v sodiu~ bicarbonate , :, .
'' ~ .~'' ' To l.O ml of the lytic diluent in a test tube i8 added with swirling 0.1 ml of whole blood anticoagulated with ~DTA. 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 60C to 70C water bath with swirling for thirty seconds. The preparation is analyzed by a COULTER TPS-l Flow Cytometer equipped with a 30 mwatt HeNe laser. Forward light scatter is measured in an angular range of 2~o 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 intenmediate fluorescence-intermediate light scatter; the neutrophils appear at intermediate fluorescence-high light scatter; 5 and the eosinophils at high fluorescence-high light scatter.
Example 3 White blood cell histogram by light Acatter vs DC volume using a square hole flow cytometer.
FORMULATIONS
Lytic diluent 0.16% wlv 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 wieh swirling 0.1 ml whole blood anticoagulated with EDTA. As ~oon as the blood solution clears, in approximaeely five ~o seven seconds, 2.0 ml of the fixing rea~ent i8 added with swirling. The solution i8 brought to isoconductivity ~ith re~pect~to the sheath fluid by addition of a suitable volume of 1.6X w/v sodium chloride to the solution.
The preparation i8 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. Fo~ward light Acatter, DC volume and RF ~ize 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 cluseers, 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 volu~e 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.
FORMnLATIONS
Lytic diluent 0.4% w/v saponin 0.2~ w/v sodium chloride 0.5~ v/v 2-phenoxyethanol 0.03% wiv 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 diluen~ in a test tube is added with swirling t~ 0.10 ml whole blood anticoagulated with EDTA. After ten seconds, 1.0 ~L
of fixing reagent is added with swirling. The mixture i8 heated in a 70C water bath for fifteen ~econds. The ~olution is brought to isoconductivity with the sheath fluid by addition of a suitable volume of 1.6% wiv sotium chloride.
The preparation i9 analyzed by the instrument descriked in Exflmple 3, maasuring opacity vs~DC volume, Foor di~tinct cluster~ of :
:
- '' :
., ~ ,, , ' white blood cells a~e found, as s~.own in FIGURE 4. Lymphocytes appear at low to high opacity-low volume; monocytes are seen at low opacity-high volume; neutrophil~ are seen at intermediate opacity-high volume and eosinophils are seen at high opacity-high volume.
5This 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.
10Example 5 White blood cell histogram by opacity (RF size/DC valume), vs DC volume using the focused square hole flow cell or an unfocused standard COULTER aperture. Demonstration of the predilution method.
Prediluent 0.3Z 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 ~odium 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 i9 added with ~wirling, and the ~ample i8 heated in a 70C water bath for fifteen ~econd~. For use with fl focused qquare hole aperture, the sample i8 diluted 1 : 1 with O.9g w/v of ~odium chloride ~nd the conductivity i8 adjusted to be equal to that of the sheath fluid by suitable addition of 2.0Z w/v of sodium chloride.
For use with an unfocused aperture in fln aperture bath, the ~ample i8 diluted 10 times with isotonic diluent. No correction for conductivity i~ needed. Determination of the opacity (RF size/DC
volume) V8 DC volume gives four Ieukocyte clu~ters, as shawn in FIGURE
5. The lymphocyteR appear at low to high opacity-low volu~e;
. ` `
: ' ` ` ' `
., .~ . .
... - ;.. ..
monocytes and neutrophilg are seen at intermediate opacity and high volume, as clearly separated populations; and eosinophils are located at high opacity and high vol~e.
Example 6 5White 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 bicarb~nate 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 ~nstrument aspirates a whole b~ood sample anticoagulated with ~DTA into the sampling loop, where 28 ~ of blood is segmented. A 170 uL portion of the lytic reagent is delivered into the sample chamber, followed promptly by ~he wbole blood 3ample carried by 150 uL of the prediluent. Mixing of the sample chamber by nutation i3 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 70C in about seven seconds. After seven more secondc at this temperature 170 uL of the diluent is added for conductivity adjustment, the mixing i8 stopped, and the sample is fed into a focused flow cell for measurement of the DC volume and RF ~ize parameters. After sampling, the bottom drain i~
opened and the exces~ sample i8 r;emoved~fro~ the sample chamber, which is further rinsed in orter to reduce "carry over", and to cool the ssmple chamber in preparation for the next sampl~e. ~ ~
Mea~uremenC of ~he DC volume and RF size~parameters allow the discrimination of four leukocyte~popul8tions a8 de~cribed ln the former examples and as shown in FIGUR5 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.
.:
.
. :: : : :
: ~ , - : ~
- ` : ,
between 7.0 and 8Ø 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/~.
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 solu~ion i8 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-4~*ge~, 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 75C for about 10 second~ 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 75C water bath wieh swirling to achieve heatin8.
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.
FO~MULATIONS
Lytic diluent 0.2~ w/v saponin 0.6% w/v sodium chloride l.OX ~epe~ Buffer Cyanine dye 1 ~ 10 5 ~ Di l-C3S03-(3) adjust pH to 7.2 wieh sodium hydroxide.
~ Fixing reagent 0.5% v/v glutaraldehyde 1,5: v/v so~ bicsrbona-e 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 ~en seconds, 2.0 ml of the fixing r~agent is added with swirling. After fifteen seconds the mixture is heated in a 70~C water bath for fifteen seconds with swirling.
The preparation i8 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 ~ass filter are placed in the fluorescence channel~ The white blood cell histogram i3 collected as fluorescence vs light scatter (2 to 20), as shown in FIGU~E 1, or as fluorescence/light scaeter V8 light scatter.
Four distinct clusters of white blood cells are observed.
Sorting of cells in each of these clus~ers and microscopic examination shows that the cells are lymphocytes at-low 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 i8 observed at low light scatter-intermediate fluorescence.
Example 2 White blood cell histogram by fluorescence and light scatter using a COULTER TPS-l equipped with 30 mwatt HeNe laser at 633 nm.
FORMULATIO~S
Lytic diluent 0.2% w/v saponin 0.5% w/v sodium ~ulfate 3.0% w/v sucrose cyanine dye 1 x 10-5 M DiIC3S03-(5) Fixîng reagent 1.0% v/v glutsraldehyde 0.8% w/v sodium chloride 0.5% w/v sodiu~ bicarbonate , :, .
'' ~ .~'' ' To l.O ml of the lytic diluent in a test tube i8 added with swirling 0.1 ml of whole blood anticoagulated with ~DTA. 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 60C to 70C water bath with swirling for thirty seconds. The preparation is analyzed by a COULTER TPS-l Flow Cytometer equipped with a 30 mwatt HeNe laser. Forward light scatter is measured in an angular range of 2~o 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 intenmediate fluorescence-intermediate light scatter; the neutrophils appear at intermediate fluorescence-high light scatter; 5 and the eosinophils at high fluorescence-high light scatter.
Example 3 White blood cell histogram by light Acatter vs DC volume using a square hole flow cytometer.
FORMULATIONS
Lytic diluent 0.16% wlv 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 wieh swirling 0.1 ml whole blood anticoagulated with EDTA. As ~oon as the blood solution clears, in approximaeely five ~o seven seconds, 2.0 ml of the fixing rea~ent i8 added with swirling. The solution i8 brought to isoconductivity ~ith re~pect~to the sheath fluid by addition of a suitable volume of 1.6X w/v sodium chloride to the solution.
The preparation i8 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. Fo~ward light Acatter, DC volume and RF ~ize 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 cluseers, 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 volu~e 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.
FORMnLATIONS
Lytic diluent 0.4% w/v saponin 0.2~ w/v sodium chloride 0.5~ v/v 2-phenoxyethanol 0.03% wiv 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 diluen~ in a test tube is added with swirling t~ 0.10 ml whole blood anticoagulated with EDTA. After ten seconds, 1.0 ~L
of fixing reagent is added with swirling. The mixture i8 heated in a 70C water bath for fifteen ~econds. The ~olution is brought to isoconductivity with the sheath fluid by addition of a suitable volume of 1.6% wiv sotium chloride.
The preparation i9 analyzed by the instrument descriked in Exflmple 3, maasuring opacity vs~DC volume, Foor di~tinct cluster~ of :
:
- '' :
., ~ ,, , ' white blood cells a~e found, as s~.own in FIGURE 4. Lymphocytes appear at low to high opacity-low volume; monocytes are seen at low opacity-high volume; neutrophil~ are seen at intermediate opacity-high volume and eosinophils are seen at high opacity-high volume.
5This 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.
10Example 5 White blood cell histogram by opacity (RF size/DC valume), vs DC volume using the focused square hole flow cell or an unfocused standard COULTER aperture. Demonstration of the predilution method.
Prediluent 0.3Z 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 ~odium 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 i9 added with ~wirling, and the ~ample i8 heated in a 70C water bath for fifteen ~econd~. For use with fl focused qquare hole aperture, the sample i8 diluted 1 : 1 with O.9g w/v of ~odium chloride ~nd the conductivity i8 adjusted to be equal to that of the sheath fluid by suitable addition of 2.0Z w/v of sodium chloride.
For use with an unfocused aperture in fln aperture bath, the ~ample i8 diluted 10 times with isotonic diluent. No correction for conductivity i~ needed. Determination of the opacity (RF size/DC
volume) V8 DC volume gives four Ieukocyte clu~ters, as shawn in FIGURE
5. The lymphocyteR appear at low to high opacity-low volu~e;
. ` `
: ' ` ` ' `
., .~ . .
... - ;.. ..
monocytes and neutrophilg are seen at intermediate opacity and high volume, as clearly separated populations; and eosinophils are located at high opacity and high vol~e.
Example 6 5White 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 bicarb~nate 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 ~nstrument aspirates a whole b~ood sample anticoagulated with ~DTA into the sampling loop, where 28 ~ of blood is segmented. A 170 uL portion of the lytic reagent is delivered into the sample chamber, followed promptly by ~he wbole blood 3ample carried by 150 uL of the prediluent. Mixing of the sample chamber by nutation i3 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 70C in about seven seconds. After seven more secondc at this temperature 170 uL of the diluent is added for conductivity adjustment, the mixing i8 stopped, and the sample is fed into a focused flow cell for measurement of the DC volume and RF ~ize parameters. After sampling, the bottom drain i~
opened and the exces~ sample i8 r;emoved~fro~ the sample chamber, which is further rinsed in orter to reduce "carry over", and to cool the ssmple chamber in preparation for the next sampl~e. ~ ~
Mea~uremenC of ~he DC volume and RF size~parameters allow the discrimination of four leukocyte~popul8tions a8 de~cribed ln the former examples and as shown in FIGUR5 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.
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.
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Claims (29)
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 comprising aqueous solutions of:
(A) a lysing reagent selected from the group consisting of a lytic diluent containing saponin, or 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.
(A) a lysing reagent selected from the group consisting of a lytic diluent containing saponin, or 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 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.
3. The reagent system of claim 3 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.
4. The reagent system of any one of claims 1, 2 or 3 in which said saponin is quillaja saponin.
5. The reagent system of any one of claims 1, 2 or 3 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 claim 1 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 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 saponic concentration in the lysing reagent of 0.15 to 0.40% w/v.
8. The reagent system of claim 1 to which has been added a water soluble preservative which inhibits the growth of microorganisms.
9. The reagent system of claim 8 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-1-oxide, and sodium azide.
10. The reagent system of claim 1 in which is included, for enhancing cell classification, at least one additive selected from the group consisting of 8 short chain alkanol substituted by phenyl or phenoxy and a polyhydroxy compound.
11. The reagent system of claim 10 wherein said additive is 2-phenoxyethanol.
12. The reagent system of claims 10 or 11 wherein said polyhydroxy compound is sucrose.
13. The reagent system of claim 1 to which a fluorescent dye has been added.
14. The reagent system of claim 13 wherein said fluorescent dye is a sulfonated cyanine dye or other sulfonated fluorescent dye.
15. The reagent system of any one of claims 1, 3 or 6 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 8Ø
16. The reagent system of claim 1 wherein said cross-linking compound is glutaraldehyde.
17. The reagent system of claim 16 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, 3 or 10 wherein the 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 lyse solution, 80 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, 10 or 16 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 being an aqueous solution of:
(A) a lysing reagent selected from the group consisting of a lytic diluent containing saponin, or a prediluent system followed by a lytic reagent 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.
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 being an aqueous solution of:
(A) a lysing reagent selected from the group consisting of a lytic diluent containing saponin, or a prediluent system followed by a lytic reagent 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 including heating said treated blood at an elevated temperature for a few seconds.
22. The method of claim 21 wherein the heating takes place at a temperature of about 60° to 75°C for about 10 to 30 seconds.
23. The method of claim 20 which further includes correlating the measured parameters of RF size/DC volume vs DC volume.
24. The method of claim 20 which further includes correlating the measured parameters of light scatter vs DC volume.
25. The method of claim 20 which further includes correlating the measured parameters of light scatter/DC volume vs DC volume.
26. The method of claim 20 which further includes correlating the measured parameters of fluorescence vs light scatter.
27. The method of claim 20 which further includes correlating of the measured parameters of fluorescence/light scatter vs light scatter.
28. The method of any one of claims 20, 23 or 26 including generating cytograms by said instrumentation, said cytograms being based on selected mathematical combinations of said primary parameters.
29. The method of any one of claims 20, 23 or 26 wherein said major categories of leukocytes are: lymphocytes, monocytes, neutrophils and eosinophils.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/615,966 US4751179A (en) | 1984-05-31 | 1984-05-31 | Method and reagents for differential determination of four populations of leukocytes in blood |
| US615,966 | 1984-05-31 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1248856A true CA1248856A (en) | 1989-01-17 |
Family
ID=24467496
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000482841A Expired CA1248856A (en) | 1984-05-31 | 1985-05-30 | Method and reagent system for four-population differential determination of leukocytes |
Country Status (9)
| Country | Link |
|---|---|
| US (1) | US4751179A (en) |
| EP (1) | EP0185048B1 (en) |
| JP (1) | JPS61502277A (en) |
| KR (1) | KR860700153A (en) |
| AU (1) | AU4401985A (en) |
| CA (1) | CA1248856A (en) |
| DE (1) | DE3587036T2 (en) |
| ES (1) | ES8609727A1 (en) |
| WO (1) | WO1985005684A1 (en) |
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- 1985-05-09 JP JP60502359A patent/JPS61502277A/en active Pending
- 1985-05-30 ES ES543657A patent/ES8609727A1/en not_active Expired
- 1985-05-30 CA CA000482841A patent/CA1248856A/en not_active Expired
-
1986
- 1986-01-30 KR KR1019860700056A patent/KR860700153A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| JPS61502277A (en) | 1986-10-09 |
| DE3587036D1 (en) | 1993-03-11 |
| DE3587036T2 (en) | 1993-09-02 |
| EP0185048A4 (en) | 1988-09-28 |
| ES8609727A1 (en) | 1986-08-16 |
| EP0185048B1 (en) | 1993-01-27 |
| AU4401985A (en) | 1985-12-31 |
| KR860700153A (en) | 1986-03-31 |
| WO1985005684A1 (en) | 1985-12-19 |
| US4751179A (en) | 1988-06-14 |
| ES543657A0 (en) | 1986-08-16 |
| EP0185048A1 (en) | 1986-06-25 |
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| MKEX | Expiry |