US3796594A - Stain coated slides for differentially staining blood - Google Patents

Stain coated slides for differentially staining blood Download PDF

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Publication number
US3796594A
US3796594A US00222654A US3796594DA US3796594A US 3796594 A US3796594 A US 3796594A US 00222654 A US00222654 A US 00222654A US 3796594D A US3796594D A US 3796594DA US 3796594 A US3796594 A US 3796594A
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United States
Prior art keywords
staining
slide
blood
stain
drop
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Expired - Lifetime
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US00222654A
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English (en)
Inventor
F Thomae
J Geating
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General Electric Co
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General Electric Co
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    • 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

Definitions

  • Basic vital dyes stain unfixed reticulocytes specifically; when a blood sample is fixed with methyl alcohol, the basic staining is diffuse. Platelet identification and white blood corpuscle differentiation are not achieved.
  • Reticulocytes may be stained in a mixture of blood and methylene blue NN in saline solution spread and dried on a slide; but platelets are somewhat obscured by precipitate formation, and in the white blood corpuscles only the cytoplasmic granules are stained.
  • a slide is coated with a mixture of methylene blue NN and cresyl violet acetate in absolute methanol, and dried rapidly.
  • a drop of blood is applied to the stained dry surface, and spread by the weight of a cover glass.
  • reticulocytes, platelets, neutrophils (with distinction of cytoplasm and nuclei), eosinophils (with distinction of cytoplasma and nuclei), and basophils (with distinction of containing granules) are all separately identifiable.
  • the slides may be prepared and stored dry until needed for use.
  • a solution of 1.0 gram of cresyl violet acetate in 25 milliliters of absolute methyl alcohol is prepared, and allowed to stand over night, i.e. from 16 to 24 hours. It is then filtered to remove any undissolved residue.
  • a drop of the dye mixture is placed at the end of a clean microscope slide.
  • Another glass slide is applied vertically on the drop and drawn broadside across the surface of the first slide. Capillary action between slide and liquid will insure even distribution of the dye. It is particularly important that the side be free of grease or fat and of particles such as dust.
  • the slide is then immediately dried by waving in air, or by a blast of clean dry gas, such as air. The slide is ready for use immediately upon drying; but it may be stored for future use at low humidity. High giimidity has been found to cause deformation of the dye
  • a sample site eg finger
  • the first drop of blood is wiped off.
  • the next drop is applied directly to the prestained surface of the slide.
  • a cover slip is immediately placed over the small drop of blood.
  • the weight of the cover slip may suflice to spread the drop uniformly over the slide; if it does not, slight pressure on the cover slip (conveniently with the clean end of a capillary tube) will cause it to spread thus.
  • the staining characteristics differ from those of the prior art Wrights stain.
  • the staining characteristics of the slide coating of our. invention are the following:
  • Neutrophils contain a finely granulated cytoplasm which stains a light purple in the fresh preparation and fades to a greenish tint in the older smear. Nuclei stain a bright purple.
  • Eosinophils exhibit a bright purple nucleus and a cytoplasm packed with large uniform orange-staining granules which fade to a greenish orange when the preparation is about twelve hours old.
  • Basophils contain smaller number of uniform-size granules than eosinophils, which stain a dark purple and often completely obscure the nucleus. With focusing, an orange tinge can be observed in the granules found at the edge of the cell, an'effective identifying feature.
  • Lymphocytes develop a purple staining nucleus surrounded by a lighter purple cytoplasm which may become as dark as the nucleus as the preparation grows old.
  • Monocytes have an afiinityfor the stain similar to lymphocytes. Distinguishing features are the larger size and the greater amount of cytoplasm of the monocytes. It has been found that the differentiation is readily learned.
  • Reticulocytes contain a reddish-purple network but do not fade even after the red cells become distorted. Nevertheless, the reticulocyte count should also be made within 4 to 5 hours after the staining period because of the fading of some of the red blood corpuscles when they die.
  • the proportion of methylene blue NN in the coating may range from approximately 91% to 94%, and that of cresyl violet acetate from approximately6% to 9% Moderate departures from these proportions are, .of course, permissible, and may be desirable if it is desired to accentuate particular constituent staining. Since it is the provision of the combination of stains to the blood sample which is essential to our invention, it is not necessary that the film of dried stains be applied to the slide; it is equally feasible to provide the dried dye film on a transparent film, which is applied over a drop of blood on a clean slide.
  • Such prestained transparent films may be packaged in individual sealed envelopes, which would automatically provide a suitably dry atmosphere for storing the dye film, and would, of course, markedly simplify the problems of packaging for shipment, and of storage.
  • the use of our invention minimizes the burden upon the clinical technician.
  • the elimination of various intermediate steps of the prior art not only reduces the time consumed in a determination but tends to a higher degree of uniformity in preparations.
  • a microscopic slide having coated thereon a mixture of stains for differentially staining blood, said mixture consisting of approximately 91% to 94% by weight of methylene blue NN and 6% to 9% by weight of cresyl violet acetate.
  • a method of differentially staining a blood sample which comprises (a) drying on a microscopic slide an alcoholic solution containing approximately 91% to 94% by weight of methylene blue NN and 6% to 9% by weight of cresyl violet acetate, and

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  • 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)
US00222654A 1970-10-30 1972-02-01 Stain coated slides for differentially staining blood Expired - Lifetime US3796594A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US8581770A 1970-10-30 1970-10-30
US22265472A 1972-02-01 1972-02-01

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US3796594A true US3796594A (en) 1974-03-12

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FR (1) FR2169989B2 (enrdf_load_stackoverflow)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3871895A (en) * 1973-06-29 1975-03-18 Technicon Instr Method for preparing smears of biological liquids
US3906120A (en) * 1970-10-30 1975-09-16 Gen Electric Method for preparing slides for blood evaluation
DE2737845A1 (de) * 1975-08-08 1978-03-02 Gen Electric Verfahren zum anfaerben und versiegeln einer biologischen probe, die an einem mikroskopischen objekttraeger haftet und luftgetrocknet ist, insbesondere einer blutprobe
US4103041A (en) * 1975-04-11 1978-07-25 Boehringer Mannheim Gmbh Process for coating slides
DE2826363A1 (de) * 1978-06-16 1979-12-20 Merck Patent Gmbh Deckfolie zur verwendung in mikroskopischen faerbeverfahren und verfahren zu ihrer herstellung
US4185085A (en) * 1976-11-09 1980-01-22 Boehringer Mannheim Gmbh Differential diagnostic sperm examination
WO2013038306A1 (en) * 2011-09-13 2013-03-21 Koninklijke Philips Electronics N.V. System and kit for preparing a cytological sample for examination
US20210181085A1 (en) * 2017-10-26 2021-06-17 Essenlix Corporation Rapid measurement of platelets

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2515966C3 (de) * 1975-04-11 1979-11-22 Boehringer Mannheim Gmbh, 6800 Mannheim Vorgefärbte Objektträger für die Blutunte rsuchung

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3906120A (en) * 1970-10-30 1975-09-16 Gen Electric Method for preparing slides for blood evaluation
US3871895A (en) * 1973-06-29 1975-03-18 Technicon Instr Method for preparing smears of biological liquids
US4103041A (en) * 1975-04-11 1978-07-25 Boehringer Mannheim Gmbh Process for coating slides
DE2737845A1 (de) * 1975-08-08 1978-03-02 Gen Electric Verfahren zum anfaerben und versiegeln einer biologischen probe, die an einem mikroskopischen objekttraeger haftet und luftgetrocknet ist, insbesondere einer blutprobe
US4185085A (en) * 1976-11-09 1980-01-22 Boehringer Mannheim Gmbh Differential diagnostic sperm examination
DE2826363A1 (de) * 1978-06-16 1979-12-20 Merck Patent Gmbh Deckfolie zur verwendung in mikroskopischen faerbeverfahren und verfahren zu ihrer herstellung
US4302480A (en) * 1978-06-16 1981-11-24 Merck Patent Gesellschaft Mit Beschrankter Haftung Thin cover sheet for use in microscopic staining and a process for its production
WO2013038306A1 (en) * 2011-09-13 2013-03-21 Koninklijke Philips Electronics N.V. System and kit for preparing a cytological sample for examination
US20210181085A1 (en) * 2017-10-26 2021-06-17 Essenlix Corporation Rapid measurement of platelets

Also Published As

Publication number Publication date
FR2169989B2 (enrdf_load_stackoverflow) 1974-02-01
FR2169989A2 (enrdf_load_stackoverflow) 1973-09-14

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