US3884579A - Method for counting blood platelets - Google Patents

Method for counting blood platelets Download PDF

Info

Publication number
US3884579A
US3884579A US369827A US36982773A US3884579A US 3884579 A US3884579 A US 3884579A US 369827 A US369827 A US 369827A US 36982773 A US36982773 A US 36982773A US 3884579 A US3884579 A US 3884579A
Authority
US
United States
Prior art keywords
platelets
surface tension
red
counting
solution
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 - Lifetime
Application number
US369827A
Inventor
Thomas Mauthner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cambridge Chemical Products Inc
Original Assignee
Cambridge Chemical Products 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 Cambridge Chemical Products Inc filed Critical Cambridge Chemical Products Inc
Priority to US369827A priority Critical patent/US3884579A/en
Application granted granted Critical
Publication of US3884579A publication Critical patent/US3884579A/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

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
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/305—Fixative compositions
    • 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.]
    • 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
    • Y10T436/108331—Preservative, buffer, anticoagulant or diluent

Definitions

  • the present invention is directed to compositions for staining the platelets, preserving them against agglutination, and most importantly eliminating the masking red and white cells, and to a method of counting platelets employing the composition.
  • the present invention is broadly directed to a composition having a surface tension sufficiently low so that when a blood sample is dissolved in the composition the large, relatively low surface tension red and white cells are quickly hemolyzed, and yet sufficiently high so that the smaller, higher surface tension platelets are not destroyed by the composition over relatively larger periods of time. Dissolving a blood sample in this composition allows platelets to be counted easily and with a high degree of accuracy with conventional microscopes, phase microscopes or electron microscopes.
  • the present invention is based on my discovery that when whole blood serum is dissolved in a composition having a surface tension, measured at 22 C in distilled water of between 31.0 and 29.8 dynes per centimeter, the red and white blood cells in the serum will be hemolyzed within a relatively few seconds, but the platelets will not be hemolyzed in long periods.
  • These surface tension limitations are relatively sharp and when a compound having a surface tension of more than 31.5 dynes per centimeter is employed the red and white cells are not fully hemolyzed within a matter of a few minutes.
  • a composition having a surface tension of less than 29.8 dynes per centimer is employed some of the platelets are also quickly hemolyzed.
  • the method of the present invention therefore comprises dissolving a blood sample in a composition having a surface tension within these critical limits and then microscopicly studying the dissolved sample after about 30 seconds to count the platelets which are readily observable in the absence of the red and white cells.
  • the action of the nonionic surfactants in hemolyzing the red and white cells is believed to be solely through a reduction of Vanderwaals forces cause a bursting of the cells.
  • the effect of the composition on the platelets is not only a function of the surface tension of the composition but also of the percentage of surfactant in the composition. While the ability of the surfactant to lower the surface tension of the composition stabilizes after a certain percentage of the surfactant in the composition has been reached increasing percentages of surfactant above the plateau percentage will tend to increase the rate which the platelets are hemolyzed. For example, a composition containing 0.05 percent of a given surfactant may have a resulting surface tension of 30.0 dynes per centimeter.
  • compositions used in connection with the preferred embodiment to the invention should not contain surfactants in concentrations greater than about 0.1 percent.
  • the preferred composition of the present invention consists of a solution, in water and alcohol of a surfactant that produces the desired surface tension in the desired concentrations and certain known preservatives as well as unique preservatives and a unique stain.
  • the surfactant is preferably present in a solution in concentrations of between 0.001 and 0.1 percent.
  • the surfactant is preferably chosen from the group consisting of nonionic, cationic and antionic surfactants which are capable of producing the desired surface tension in the percentages indicated.
  • Surfactants which would produce the desired surface tension in concentrations lower than 0.001 percent but would produce lower surface tensions in higher concentrations are undesirable since small variations in concentration of these surfactants may produce appreciable variations on the surface tension of the composition.
  • Nonionic surfactants producing the desired surface tension in the desired concentration are preferable to those antionic and cationic surfactants which will produce the surface tension since the later tend to chemically react with various of the blood constituents to produce products which complicate the counting process.
  • nonionic, anionic. cationic surfactants may be used in connection with the compositions of the present invention. While ionic surfactants have the capability of producing the desired range of surface tension in low concentrations and provide the advantage of a clear background for counting the platelets, they have two disadvantages. First they react with protein in the blood forming complex salts which may obscure the counting process and secondly they have a tendency to hemolyze the platelets at relatively high surface tensions relative to the nonionic surfactants. This is probably due to chemical attack on the platelets by the polar groups of the ionic surfactants whereas the nonionic surfactants tend to hemolyze the red and white cells solely due to a reduction in Vanderwaals forces.
  • a preferred composition also contains potassium fluoride to prevent the rapid destruction of the sample supported on a slide by exposure to airborne bacteria.
  • Potassium fluoride being a salt of a strong acid and a strong base does not have a PH effect on the solution.
  • the fluoride ion provided by the potassium fluoride is the active component in the preservative action.
  • the high solubility of the potassium fluoride is extremely useful in preserving the blood samples on the slide.
  • the preferred composition includes formaldehyde as a solution preservative and a chelating agent such as sodium citrate to stop coagulation.
  • the preferred solution further contains alphazurine 2G dye.
  • This particular dye has not previously been used for the dying of platelets and I have found that it is more selective and provides a better contrast with the background products than brilliant cresyl blue which has previously been used as a stain for platelets.
  • the preferred composition also contains an alkaline metal oxalate. and preferably potassium oxalate, which acts as an anticoagulant and a chelating agent to improve the clarity of observation of the stained platelets.
  • my invention greatly simplifies and increases the accuracy of microscopic counting of blood platelets using a relatively low cost and highly stable solution.
  • This composition has a surface tension 30.2 dyn/cm. As set forth above, the red and white blood cells are completely hemolyzed within 2 minutes of dissolving a blood sample in the solution. A blood sample dissolved in this preferred composition and then analyzed by microscope tested out to have a platelet count within 1.4 percent of the same count achieved using a Coulter electronic counter.
  • EXAMPLE 2 EXAMPLE 3
  • the basic formula of Example 1 is used but the nonionic surfactant was replaced with a cationic surfactant, alkyl dimethyl benzyl ammonium chloride in the amount of 0.004 percent by weight of the solution.
  • the solution produced a surface tension of 29.8 dyn/cm., the red and white cells were completely hemolyzed within 2 /2 minutes and the microscopic count of platelets in the solution was within 2.4 percent of the count as analyzed by a Coulter counter.
  • EXAMPLE 4 Various combinations of ionic and nonionic surfactants may also be employed with the compositions of the present invention. When ionic surfactants are added to the composition containing nonionic surfactants a clearer background is attained.
  • One such composition involves the base composition of Example I but employing 0.006 percent by weight of the nonionic alkyl phenoxy (polyethylene oxy) ethanol and 0.002 percent by weight of anionic Sodium Lauryl Sulfate. This composition produced a surface tension of 30.1 dyn/cm., completely hemolyzed the red and white blood cells in less than four minutes and produced a platelet count under microscope of within 1.6 percent of that achieved with the Coulter counter.
  • EXAMPLE 5 In this example 0.005 percent of the nonionic surfactant of Example 1 was combined with a 0.002 percent of the cationic surfactant of Example 3. The resultant composition had a surface tension of 30.1 dyn/cm., completely hemolyzed the red and white cells within four minutes and provided a count under microscopic analysis of within 2 percent of the count achieved with the Coulter counter.
  • the method of counting platelets in a specimen of whole blood serum comprising dissolving the specimen in a solution having a surface tension in distilled water. measured at 22C of between 29.8 and 31.0 dynes/cm. and observing and counting the platelets through a microscope after the red and white cells have been hemolyzed.
  • the method of counting the blood platelets in a sample of whole blood serum comprising dissolving the sample in a solution containing a surfactant. the solution having a surface tension in distilled water, measured at 22 C. of between 29.8 and 31.0 dynes/cm. to hemolyze the red and white cells, and observing and counting the platelets as viewed with a microscope.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Ecology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

To facilitate the direct observation and counting of platelets in whole blood serum, a blood sample is mixed with a solution which hemolyzes the red and white cells in a short time without destroying any of the platelets. The platelets can then be observed and counted through a microscope without being masked by the larger cells. The composition includes a dye, preservatives and fixatives and a surfactant which lowers the surface tension of the solution to a controlled level which hemolyzes the red and white cells while leaving the higher surface tension platelets undisturbed.

Description

United States Patent Mauthner [451 May 20, 1975 METHOD FOR COUNTING BLOOD PLATELETS Thomas Mauthner, Livonia, Mich.
Cambridge Chemical Products, Inc., Detroit, Mich.
Filed: June 14, 1973 Appl. No.: 369,827
Inventor:
Assignee:
US. Cl. 356/39; 252/408; 356/36 Int. Cl G0ln 33/16; G0ln l/OO Field of Search 356/36, 38, 39
References Cited UNITED STATES PATENTS 3/1970 Preston, Jr. et al 356/39 5/1973 Coulter et al 356/39 Primary Examiner-Vincent P. McGraw Attorney, Agent, or Firm-Krass & Young [5 7] ABSTRACT To facilitate the direct observation and counting of platelets in whole blood serum, a blood sample is mixed with a solution which hemolyzes the red and white cells in a short time without destroying any of the platelets. The platelets can then be observed and- 2 Claims, No Drawings METHOD FOR COUNTING BLOOD PLATELETS BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to methods of treating whole blood serum so as to hemolyze the red and white cells without disturbing the platelets to facilitate micro scopic examination and counting of the platelets and to compositions for use in such treatment.
2. Prior Art The count of platelets per unit volume of whole blood serum is an important diagnostic indicator, particularly in connection with disorders of blood coagulation. Electronic devices such as the Coulter counter are available for counting the platelets in a blood sample, but with high accuracy are expensive and are often not available in a modest clinical laboratory. Microscopic techniques for counting blood platelets are handicapped by the fact that the platelets are much smaller than the accompanying red and white blood cells and these larger cells mask some of the platelets making it extremely difficult to obtain an accurate platelet count.
The problem of counting platelets is further complicated by the fact that the platelets are colorless and the tendency of the platelets to agglutinate, disentegrate readily, and react with airborne bacteria. Previous platelet stain solutions have been prepared using formaldehyde, sodium citrate and brilliant cresyl blue dye. These solutions have been useful in making rough estimates of the platelet count but no microscopic method has been previously regarded as satisfactory.
The present invention is directed to compositions for staining the platelets, preserving them against agglutination, and most importantly eliminating the masking red and white cells, and to a method of counting platelets employing the composition.
SUMMARY OF THE INVENTION The present invention is broadly directed to a composition having a surface tension sufficiently low so that when a blood sample is dissolved in the composition the large, relatively low surface tension red and white cells are quickly hemolyzed, and yet sufficiently high so that the smaller, higher surface tension platelets are not destroyed by the composition over relatively larger periods of time. Dissolving a blood sample in this composition allows platelets to be counted easily and with a high degree of accuracy with conventional microscopes, phase microscopes or electron microscopes.
The present invention is based on my discovery that when whole blood serum is dissolved in a composition having a surface tension, measured at 22 C in distilled water of between 31.0 and 29.8 dynes per centimeter, the red and white blood cells in the serum will be hemolyzed within a relatively few seconds, but the platelets will not be hemolyzed in long periods. These surface tension limitations are relatively sharp and when a compound having a surface tension of more than 31.5 dynes per centimeter is employed the red and white cells are not fully hemolyzed within a matter of a few minutes. When a composition having a surface tension of less than 29.8 dynes per centimer is employed some of the platelets are also quickly hemolyzed. The method of the present invention therefore comprises dissolving a blood sample in a composition having a surface tension within these critical limits and then microscopicly studying the dissolved sample after about 30 seconds to count the platelets which are readily observable in the absence of the red and white cells.
The action of the nonionic surfactants in hemolyzing the red and white cells is believed to be solely through a reduction of Vanderwaals forces cause a bursting of the cells. The effect of the composition on the platelets is not only a function of the surface tension of the composition but also of the percentage of surfactant in the composition. While the ability of the surfactant to lower the surface tension of the composition stabilizes after a certain percentage of the surfactant in the composition has been reached increasing percentages of surfactant above the plateau percentage will tend to increase the rate which the platelets are hemolyzed. For example, a composition containing 0.05 percent of a given surfactant may have a resulting surface tension of 30.0 dynes per centimeter. Concentration of 0.25 percent of the same surfactant may only lower the surface tension to 29.8 dynes per centimeter but will still greatly accelerate the rate at which platelets in the blood serum are hemolyzed. When the percentage of surfactant is so high that any platelets are hemolyzed in less than about 5 minutes the composition becomes extremely difficult to use in connection with the method of the present invention. Accordingly, compositions used in connection with the preferred embodiment to the invention should not contain surfactants in concentrations greater than about 0.1 percent.
The preferred composition of the present invention consists of a solution, in water and alcohol of a surfactant that produces the desired surface tension in the desired concentrations and certain known preservatives as well as unique preservatives and a unique stain. The surfactant is preferably present in a solution in concentrations of between 0.001 and 0.1 percent. The surfactant is preferably chosen from the group consisting of nonionic, cationic and antionic surfactants which are capable of producing the desired surface tension in the percentages indicated. Surfactants which would produce the desired surface tension in concentrations lower than 0.001 percent but would produce lower surface tensions in higher concentrations are undesirable since small variations in concentration of these surfactants may produce appreciable variations on the surface tension of the composition. Nonionic surfactants producing the desired surface tension in the desired concentration are preferable to those antionic and cationic surfactants which will produce the surface tension since the later tend to chemically react with various of the blood constituents to produce products which complicate the counting process.
Various nonionic, anionic. cationic surfactants may be used in connection with the compositions of the present invention. While ionic surfactants have the capability of producing the desired range of surface tension in low concentrations and provide the advantage of a clear background for counting the platelets, they have two disadvantages. First they react with protein in the blood forming complex salts which may obscure the counting process and secondly they have a tendency to hemolyze the platelets at relatively high surface tensions relative to the nonionic surfactants. This is probably due to chemical attack on the platelets by the polar groups of the ionic surfactants whereas the nonionic surfactants tend to hemolyze the red and white cells solely due to a reduction in Vanderwaals forces.
A preferred composition also contains potassium fluoride to prevent the rapid destruction of the sample supported on a slide by exposure to airborne bacteria. Potassium fluoride. being a salt of a strong acid and a strong base does not have a PH effect on the solution. The fluoride ion provided by the potassium fluoride is the active component in the preservative action. The high solubility of the potassium fluoride is extremely useful in preserving the blood samples on the slide.
ln addition to the potassium fluoride the preferred composition includes formaldehyde as a solution preservative and a chelating agent such as sodium citrate to stop coagulation.
The preferred solution further contains alphazurine 2G dye. This particular dye has not previously been used for the dying of platelets and I have found that it is more selective and provides a better contrast with the background products than brilliant cresyl blue which has previously been used as a stain for platelets. The preferred composition also contains an alkaline metal oxalate. and preferably potassium oxalate, which acts as an anticoagulant and a chelating agent to improve the clarity of observation of the stained platelets.
Alphazurine 2G dye 0.125% by weight Distilled water and Surfactant Balance Varying percentages of surfactants to be tested between 0.002 and 0.01 percent were added to the base solution. Slides prepared with blood samples dissolved in the solution were observed to determine the time required to achieve complete hemolysis of the red and white cells. In those cases where the time of complete hemolysis was less than about 20 minutes, the count of the platelets were made using the solution and microscopic techniques and the results were compared to the platelet count as determined by a Coulter electronic counter to determine if any appreciable percentage of the platelets were destroyed. The surface tensions of the various solutions were also measured using a Fisher Du Nuoy Tensiometer, Model 21. Using this method, the following results were achieved:
Percentage Time of Complete Microscopic Platelet Count Surface Tension At Added to Red and White Relation to Count Made 22c Fisher DuNuoy Surfactant Formula Cell Hemolysis by Coulter Electronic Counter Tensiometer Model 21 9-10 MOL Alkyl Phenoxy (poly 003% over 120 min. Not Performed 32.7 ethylene oxy) .0057: over 80 min. -3.6% 31.6 Ethanol 008% 8 min. l.87( 30.6 009% 6 min. 2.0% 30.5 01% 1.8 min. -l.. 7z 30.2 Sodium Alkyl Ether .0027: over 200 min. Not Performed 32.8 Sulfate .0049? 1 min. Platelets Destroyed 29.7 .()08% 1 min. Platelets Destroyed 28.9 15-16 MOL Alkyl Phenoxy (poly- .00371 240 min. Not Performed 34.4 ethylene oxy) .00772 240 min. Not Performed 33.5 Ethanol 015% min. Platelets Destroyed 33.5 Sodium Lauryl .0037: min. 8.4% 32.0 Sulphate 005% 20 min. .8% 31.2 .017: 1 min. Platelets Destroyed 29.2 Alkyl Dimethyl 002% 80 min. 4.2% 32.2 Benzyl Ammonium 004% 2.5 min. 2.1% 29.8 Chloride 006% 1 min. Platelets Destroyed 26.2 Alkyl Aryl Sulfonate .0047! 200 min. Not Performed 33.1 008% 200 min. Not Performed 32.0 015% 1 min. Platelets Destroyed 29.4 Sodium Di-octyl 002% over 200 min. Not Performed 32.8 Sulfo Succinate 004% 1 min. Platelets Destroyed 27.6 006% 1 min. Platelets Destroyed 23.4 Substituted 03% 200 min. Not Performed 34.6 lmidazoline .0171 200 min. Not Performed 31.6 01% 1 min. Platelets Destroyed 31.2
In the preferred practice of the present invention a The following preferred examples of my invention small quantity of blood sample is introduced into apare intended to be illustrative only and not to limit the proximately 100 times that volume of the solution. The two are thoroughly mixed and spread on a slide. In about one minute after the slide preparation it is viewed in a microscope and the stained platelets are fully visible without interference from the red and white cells which have been hemolyzed.
It is therefore seen that my invention greatly simplifies and increases the accuracy of microscopic counting of blood platelets using a relatively low cost and highly stable solution.
To determine the utility of various surfactants in connection with the present invention, the following base composition was prepared:
scope of the invention:
This composition has a surface tension 30.2 dyn/cm. As set forth above, the red and white blood cells are completely hemolyzed within 2 minutes of dissolving a blood sample in the solution. A blood sample dissolved in this preferred composition and then analyzed by microscope tested out to have a platelet count within 1.4 percent of the same count achieved using a Coulter electronic counter.
EXAMPLE 2 EXAMPLE 3 In this example the basic formula of Example 1 is used but the nonionic surfactant was replaced with a cationic surfactant, alkyl dimethyl benzyl ammonium chloride in the amount of 0.004 percent by weight of the solution. The solution produced a surface tension of 29.8 dyn/cm., the red and white cells were completely hemolyzed within 2 /2 minutes and the microscopic count of platelets in the solution was within 2.4 percent of the count as analyzed by a Coulter counter.
EXAMPLE 4 Various combinations of ionic and nonionic surfactants may also be employed with the compositions of the present invention. When ionic surfactants are added to the composition containing nonionic surfactants a clearer background is attained.
One such composition involves the base composition of Example I but employing 0.006 percent by weight of the nonionic alkyl phenoxy (polyethylene oxy) ethanol and 0.002 percent by weight of anionic Sodium Lauryl Sulfate. This composition produced a surface tension of 30.1 dyn/cm., completely hemolyzed the red and white blood cells in less than four minutes and produced a platelet count under microscope of within 1.6 percent of that achieved with the Coulter counter.
EXAMPLE 5 In this example 0.005 percent of the nonionic surfactant of Example 1 was combined with a 0.002 percent of the cationic surfactant of Example 3. The resultant composition had a surface tension of 30.1 dyn/cm., completely hemolyzed the red and white cells within four minutes and provided a count under microscopic analysis of within 2 percent of the count achieved with the Coulter counter.
Having thus described by invention, I claim:
1. The method of counting platelets in a specimen of whole blood serum comprising dissolving the specimen in a solution having a surface tension in distilled water. measured at 22C of between 29.8 and 31.0 dynes/cm. and observing and counting the platelets through a microscope after the red and white cells have been hemolyzed.
2. The method of counting the blood platelets in a sample of whole blood serum comprising dissolving the sample in a solution containing a surfactant. the solution having a surface tension in distilled water, measured at 22 C. of between 29.8 and 31.0 dynes/cm. to hemolyze the red and white cells, and observing and counting the platelets as viewed with a microscope.

Claims (2)

1. The method of counting platelets in a specimen of whole blood serum comprising dissolving the specimen in a solution having a surface tension in distilled water, measured at 22*C of between 29.8 and 31.0 dynes/cm, and observing and counting the platelets through a microscope after the red and white cells have been hemolyzed.
2. The method of counting the blood platelets in a sample of whole blood serum comprising dissolving the sample in a solution containing a surfactant, the solution having a surface tension in distilled water, measured at 22* C. of between 29.8 and 31.0 dynes/cm. to hemolyze the red and white cells, and observing and counting the platelets as viewed with a microscope.
US369827A 1973-06-14 1973-06-14 Method for counting blood platelets Expired - Lifetime US3884579A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US369827A US3884579A (en) 1973-06-14 1973-06-14 Method for counting blood platelets

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US369827A US3884579A (en) 1973-06-14 1973-06-14 Method for counting blood platelets

Publications (1)

Publication Number Publication Date
US3884579A true US3884579A (en) 1975-05-20

Family

ID=23457098

Family Applications (1)

Application Number Title Priority Date Filing Date
US369827A Expired - Lifetime US3884579A (en) 1973-06-14 1973-06-14 Method for counting blood platelets

Country Status (1)

Country Link
US (1) US3884579A (en)

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2830524A1 (en) * 1977-07-21 1979-02-01 Technicon Instr METHOD FOR PREPARING A CELL SUSPENSION FROM BLOOD TO DIFFERENTIATE WHITE BODY CELLULARS AND BLOOD PLATES FROM OTHER BLOOD PARTICLES
US4160644A (en) * 1977-06-13 1979-07-10 Streck Laboratories, Inc. Platelet reference control and method of preparation
US4198206A (en) * 1977-06-13 1980-04-15 Ryan Wayne L Method for preparing a platelet reference control
US4206077A (en) * 1977-10-07 1980-06-03 Djuro Rodjak Agent for facilitating the counting of thrombocytes in blood samples
FR2441172A1 (en) * 1978-11-10 1980-06-06 Ryan Wayne Stable blood platelet reference sample - obtd. by addition of a solid polyethylene glycol to the suspension of platelets or to a diluent for platelets
JPS55142248A (en) * 1979-04-24 1980-11-06 Toa Medical Electronics Co Ltd Diluting liquid for blood platelet counting and blood platelet counting method using the same
US4250051A (en) * 1978-12-26 1981-02-10 Coulter Electronics, Inc. Preservative for use in calibrator compositions for blood analysis
US4287087A (en) * 1977-02-25 1981-09-01 Research Triangle Institute Fixed-dried blood platelets
US4302355A (en) * 1977-08-01 1981-11-24 Warner-Lambert Company Platelet reference control
US4455376A (en) * 1979-09-17 1984-06-19 R. J. Harvey Instrument Corp. Photometric methods for counting the particulate components of blood
WO1985005684A1 (en) * 1984-05-31 1985-12-19 Coulter Electronics, Inc. Method and reagent system for four-population differential determination of leukocytes
US4801549A (en) * 1985-09-06 1989-01-31 Technicon Instruments Corporation Method for the determination of a differential white blood cell count
WO1989001048A1 (en) * 1987-08-06 1989-02-09 Streck Laboratories, Inc. Platelet aggregation reagent, reagent container and method of determining platelet aggregation in edta-anticoagulated blood
US4978624A (en) * 1985-09-06 1990-12-18 Technicon Instruments Corporation Reagent for the determination of a differential white blood cell count
US5039487A (en) * 1987-12-22 1991-08-13 Board Of Regents, The University Of Texas System Methods for quantifying components in liquid samples
US5116539A (en) * 1988-01-27 1992-05-26 Toa Medical Electronics Co., Ltd. Reagent and method for measuring leukocytes and hemoglobin in blood
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
WO1997037229A1 (en) * 1996-03-29 1997-10-09 University Of British Columbia Platelet count assay using platelet granule proteins
EP0955543A1 (en) * 1998-05-07 1999-11-10 Immunotech S.A. New reagents and methods for erythrocyte lysis
US20080102526A1 (en) * 2006-10-30 2008-05-01 Sysmex Corporation Reagent, reagent kit and analyzing method
CN101173921B (en) * 2006-10-30 2011-11-09 希森美康株式会社 Reagent, reagent kit and analyzing method

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3503684A (en) * 1966-11-09 1970-03-31 Perkin Elmer Corp Method and apparatus for detecting mitotic blood cells on a blood cell sample slide
US3733548A (en) * 1971-04-28 1973-05-15 Coulter Electronics Apparatus and method for measuring particle concentration of a suspension passing through a sensing zone

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3503684A (en) * 1966-11-09 1970-03-31 Perkin Elmer Corp Method and apparatus for detecting mitotic blood cells on a blood cell sample slide
US3733548A (en) * 1971-04-28 1973-05-15 Coulter Electronics Apparatus and method for measuring particle concentration of a suspension passing through a sensing zone

Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4287087A (en) * 1977-02-25 1981-09-01 Research Triangle Institute Fixed-dried blood platelets
US4160644A (en) * 1977-06-13 1979-07-10 Streck Laboratories, Inc. Platelet reference control and method of preparation
US4198206A (en) * 1977-06-13 1980-04-15 Ryan Wayne L Method for preparing a platelet reference control
DE2830524A1 (en) * 1977-07-21 1979-02-01 Technicon Instr METHOD FOR PREPARING A CELL SUSPENSION FROM BLOOD TO DIFFERENTIATE WHITE BODY CELLULARS AND BLOOD PLATES FROM OTHER BLOOD PARTICLES
US4302355A (en) * 1977-08-01 1981-11-24 Warner-Lambert Company Platelet reference control
US4206077A (en) * 1977-10-07 1980-06-03 Djuro Rodjak Agent for facilitating the counting of thrombocytes in blood samples
FR2441172A1 (en) * 1978-11-10 1980-06-06 Ryan Wayne Stable blood platelet reference sample - obtd. by addition of a solid polyethylene glycol to the suspension of platelets or to a diluent for platelets
US4250051A (en) * 1978-12-26 1981-02-10 Coulter Electronics, Inc. Preservative for use in calibrator compositions for blood analysis
JPH0225150B2 (en) * 1979-04-24 1990-05-31 Toa Medical Electronics
JPS55142248A (en) * 1979-04-24 1980-11-06 Toa Medical Electronics Co Ltd Diluting liquid for blood platelet counting and blood platelet counting method using the same
US4455376A (en) * 1979-09-17 1984-06-19 R. J. Harvey Instrument Corp. Photometric methods for counting the particulate components of blood
WO1985005684A1 (en) * 1984-05-31 1985-12-19 Coulter Electronics, Inc. Method and reagent system for four-population differential determination of leukocytes
US4751179A (en) * 1984-05-31 1988-06-14 Coulter Electronics, Inc. Method and reagents for differential determination of four populations of leukocytes in blood
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
US4801549A (en) * 1985-09-06 1989-01-31 Technicon Instruments Corporation Method for the determination of a differential white blood cell count
US4978624A (en) * 1985-09-06 1990-12-18 Technicon Instruments Corporation Reagent for the determination of a differential white blood cell count
WO1989001048A1 (en) * 1987-08-06 1989-02-09 Streck Laboratories, Inc. Platelet aggregation reagent, reagent container and method of determining platelet aggregation in edta-anticoagulated blood
US5039487A (en) * 1987-12-22 1991-08-13 Board Of Regents, The University Of Texas System Methods for quantifying components in liquid samples
US5116539A (en) * 1988-01-27 1992-05-26 Toa Medical Electronics Co., Ltd. Reagent and method for measuring leukocytes and hemoglobin in blood
WO1997037229A1 (en) * 1996-03-29 1997-10-09 University Of British Columbia Platelet count assay using platelet granule proteins
US6027904A (en) * 1996-03-29 2000-02-22 University Of British Columbia Platelet count assay using thrombospondin or β-thromboglobulin
EP0955543A1 (en) * 1998-05-07 1999-11-10 Immunotech S.A. New reagents and methods for erythrocyte lysis
FR2778413A1 (en) * 1998-05-07 1999-11-12 Immunotech Sa NOVEL REAGENTS AND METHOD FOR LYSIS OF ERYTHROCYTES
US6143567A (en) * 1998-05-07 2000-11-07 Immunotech Reagents and a method for the lysis of erythrocytes
AU747910B2 (en) * 1998-05-07 2002-05-30 Immunotech New reagents and a method for the lysis of erythrocytes
US20080102526A1 (en) * 2006-10-30 2008-05-01 Sysmex Corporation Reagent, reagent kit and analyzing method
EP1918709A1 (en) * 2006-10-30 2008-05-07 Sysmex Corporation Reagent, reagent kit and analyzing method
CN101173921B (en) * 2006-10-30 2011-11-09 希森美康株式会社 Reagent, reagent kit and analyzing method
US8293536B2 (en) 2006-10-30 2012-10-23 Sysmex Corporation Reagent, reagent kit and analyzing method
US8597952B2 (en) 2006-10-30 2013-12-03 Sysmex Corporation Reagent, reagent kit and analyzing method

Similar Documents

Publication Publication Date Title
US3884579A (en) Method for counting blood platelets
JP2711786B2 (en) Reagent composition and its use for cell sphering
US5731206A (en) Method and reagent system for isolation, identification and/or analysis of leukocytes from whole blood samples
US5958776A (en) Method for classifying and counting immature leukocytes
DE69535413T2 (en) Reagent and method for analyzing solid components in urine
US6664110B1 (en) Erythroblast diagnostic flow-cytometry method and reagents
DE69623174T2 (en) Method and reagent composition for performing differentiating leukocyte counts in fresh and aged whole blood samples, based on the already existing peroxidase activity of the leukocytes
US4346018A (en) Multi-purpose blood diluent and lysing agent for differential determination of lymphoid-myeloid population of leukocytes
DE69123519T2 (en) Method and device for determining ions
JP3039594B2 (en) Staining reagent and method of use
Bloom et al. The localization of lipids in human blood and bone marrow cells
US4978624A (en) Reagent for the determination of a differential white blood cell count
JPH06180314A (en) Reagent composition and identification of reticulocyte in whole blood of said composition and usage for displaying characteristic
AU602129B2 (en) Method for the determination of a differential white blood cell count
JP2007532875A (en) Reference control containing nucleated red blood cell components
US3632735A (en) Method of stabilizing fowl red blood cells for use in hematology control compositions
US3906120A (en) Method for preparing slides for blood evaluation
DE3854326T2 (en) TREATMENT TECHNOLOGY FOR WHOLE BLOOD PATTERN.
US3873272A (en) Reagent and method for albumin determination
US4575490A (en) One step method for sphering and fixing whole blood erythrocytes
Shorr A New Technic for Staining Vaginal Smears: II
US4090977A (en) Osmotically balanced anticoaglant
EP1058114A2 (en) Diluting reagent and method providing time-independent consistency in mean corpuscular volume assays
Blaies et al. A simplified method for staining mast cells with astra blue
US3553311A (en) Method of producing biological stains and reagents for biochemical determinations using ultrasonic energy