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WO1986001293A1 - Differenciation, identification et enumeration de neutrophiles, d'erythroblastes et de sous-populations de lymphocytes avec du bleu basique 141 - Google Patents

Differenciation, identification et enumeration de neutrophiles, d'erythroblastes et de sous-populations de lymphocytes avec du bleu basique 141 Download PDF

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Publication number
WO1986001293A1
WO1986001293A1 PCT/EP1985/000379 EP8500379W WO8601293A1 WO 1986001293 A1 WO1986001293 A1 WO 1986001293A1 EP 8500379 W EP8500379 W EP 8500379W WO 8601293 A1 WO8601293 A1 WO 8601293A1
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cells
blue
neutrophils
composition
mature
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Lawrence Kass
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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
    • 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

Definitions

  • the invention herein disclosed is based upon the discovery of a single dyestuff which has a staining function specific to three classes of human blood cells and which identifies, differentiates and makes possible enumeration of each of the classes.
  • mature neutrophils alone of all leukocytes are stained a bright red-violet color, while immature leukocytes of all types stain pale blue.
  • both mature and immature erythroblasts are distinguished from the first class as well as from each other and all other known cells that might be present in human biopsy specimens by a different color spectra, namely, a distinctive copper color.
  • the various sub-populations of lymphocytes B cell, T helper cell, T suppressor cell and Natural Killer cell
  • Dye staining serves as a means of discernment of otherwise indiscernable detail by distinctive color reactions on cells and their associated stainable components; metabolic; functional and/or pathological.
  • Research work involving microscopically small cells originally has been accomplished primarily by the use of the microscope in conjunction with selected energy sources including both white and fluorescent light. More recently, however, the monochromatic light made available by the development of lasers has increased. the scope of means for examination of variously dyed biological specimens for medically related ends and has brought advanced high technology means into existance and availability.
  • neutrophils have been known for many years as important factors in inflammatory response. For example, increased numbers of neutrophils are found in areas of bacterial inflammation, such as pus or infected urine. Conversely, decreased numbers of neutrophils in the blood leads to an increased risk of infections.
  • neutrophils actively phagocytize foreign particles, including bacteria, and that the granules in neutrophils contain enzymes that are capable of destroying microorganisms.
  • the laboratory technique often first used to detect infection in a suspected abcess or in the urine prior to specific microbiological verification and identification of an invading organism is the determination of increased numbers of neutrophils in a biopsy specimen of fluid, or tissues from affected areas of the body.
  • Prior art discloses a number of ways of identifying increased numbers of neutrophils in a specimen.
  • neutrophils can be identified on fixed stained smears of the fluid or tissue in question.
  • other types of blood cells such as eosinophils, lymphocytes, monocytes and basophils are also stained by these types of panoptic reagents.
  • Another method involves the direct examination of an unstained wet mount under the microscope, and presumptive identification of neutrophils in a specimen by means of their shape, size and nuclear configuration.
  • identification of these cells in an unstained specimen is subject to inaccuracies, particularly mis-identification of neutrophils as other types of leukocytes.
  • cytochemical stains Using a stain for myeloperoxidase activity, neutrophils can be identified in specimens as a result of their distinctive staining properties of their granules.
  • other types of immature neutrophils such as promyelocytes, myelocytes, metamyelocytes and bands usually contain perioxidase activity as well, so the stain is not selective for neutrophils alone.
  • the test for myeloperoxidase requires fixation, and as it is usually performed requires two or more cytochemical reagents. Some of these (benzidines, etc.) have been implicated as possible carcinogens.
  • Neutrophils are also identified in urine specimens by the use of supravital stains as formulated by Stemheimer and Malbin using crystal violet acetate and safranin which characteristically stain the granules and nuclei of these cells.
  • these dyes in combination as formulated are not specific only to mature neutrophils, since, other developmental stages of neutrophilic granulocytes, as well as othr kinds of leukocytes and nonhematopoietic particles, like casts, are stained by these dyes. Stain selectivity is lacking and specific neutrophil enumeration can be difficult.
  • Basic blue 141 also known as Basacryl Blue X-7GFL
  • Basacryl Blue X-7GFL Basacryl Blue X-7GFL
  • erythrocytes which have no nucleus, and their precursor erythroblasts including proerythroblasts and intermediate normoblasts both of which have coppery-red nuclei and blue cytoplasm, the normoblasts of smaller area (or volume).
  • FAA formalin-acetone-acetic acid
  • Plate II illustrates a black and white concept of these cells which can here be verbally described only in association with the Plate as to approximate and descriptive color of the graphic identifying cell pattern.
  • An extensive computer data base and literature search with respect to basic blue dye 141 does not suggest uses for this dyestuff other than as a textile dye except in a published paper dealing with this dye along with several hundred other dyes in wastewater sewage treatment. No suggestion has been found in the literature as to any properties and uses of basic blue 141 as a specific stain for neutrophils, or for mature and immature erythroblasts, or for the various subpopulations of lymphocytes.
  • This invention introduces and teaches direct and selective staining of neutrophils, erythroblasts, and sub-populations of lymphocytes through the use of one specific dyestuff, namely, basic blue 141.
  • Basic blue 141 is a basic, cationic dye containing an oxazi ⁇ e chroraophoric group. It is listed in the Colour Index as a textile dye, but its chemical structure has not been specifically shown. From an extended experience with various lots of the dye, basic blue 141, some variations in response has been noted. Some lots are useful directly in aqueous solutions. Where response has been at variation, adjustment of the pH level of the lot to between 5.2 to 5.5 has been corrective. Plate I identifies the compound on the basis of its characteristic spectrophotometric pattern, and is relied upon as the "finger print" of the sonamed compound.
  • a 1% aqueous distilled water solution of the powdered dye is prepared and filtered.
  • the aqueous solution of the dye is applied directly to the surface of the glass slides coated with blood, buffy coat, pus, urine sediment or other biopsy fluids or tissues containing blood cells including bone marrow aspirated from the patient.
  • a staining jar can be used.
  • the stain is effective using partially denatured biopsy specimens that have been fixed for five minutes in absolute methanol or FAA fixative, coloration of the neutrophils is particularly intense and specific using dried, unfixed specimens.
  • the cationic dye may also act as a fixative, since the cells do not wash off a prepared slide to any substantial degree after staining and drying.
  • a golden sheen develops on the surface of the liquid film on the slide after about 30 seconds exposure.
  • the biopsy coating on the slides contains primarily neutrophils
  • the golden sheen spreads evenly over the surface of the aqueous liquid dye surface.
  • the sheen may appear particulate and dispersed. After an effective time interval of contact of about five minutes, the slides are washed in distilled water until no further bleeding of the dye into the water is detected.
  • the slides are air-dried and further examined under white light absorbance under the light microscope.
  • Coverslips should be mounted on the slide with the use of immersion oil rather than mounting media containing xylene (e.g., Permount), since the reaction product in neutrophils dissolves rapidly in these materials containing organic fixatives, and eventually disappears.
  • xylene e.g., Permount
  • granules of neutrophils stain bright reddish-purple. Nuclei of neutrophils show a purple outline, and pale blue staining of nuclear chromatin.
  • Granules and cytoplasm of other leukocytes such as eosinophils, basophils and monocytes stain pale blue as do platelets.
  • nuclear chromatin of mature and immature erythroblasts stained a distinctive bright copper color, with exceptionally clear delineation of the nuclear membranes and nuclear chromatin pattern.
  • Nuclei of all other marrow cells stained pale blue.
  • Mature erythrocytes and cytoplasm of mature erythroblasts stained a grayish-copper color, whereas the cytoplasm of proerythroblasts stained deep blue. Used in this fashion, erythroblasts at all stages of maturation were made immediately visible in bone marrow specimens, facilitating their quantitation in the process of bone marrow Interpretation.
  • the shelf life of the dye basic blue 141 is indefinite.
  • the staining qualities of the dye remain stable and unaffected at normal room temperatures for at least one year of shelf life. Attention is directed to Plate I showing the spectral response curve of basic blue 141, identified sometimes under the name of Basacryl Blue X-7GFL. (This dye is available and may be supplied through BASF-Wyandotte U.S.A.)
  • the curve of Plate I is relied upon for identification of the essential dye in the absence of well-established, known chemical structure. Selective and specific staining of neutrophils and erythroblasts by basic blue 141 is useful in medical diagnosis because:
  • Basic blue 141 quickly and specifically detects the presence of neutrophils, or inflammatory cells, in specimens, making the diagnosis of infection more rapid than before.
  • Basic blue 141 complements traditional panoptic and cytochemical stains for granulocytic cells by being specific for neutrophils alone among leukocytes, thereby permitting their quantitation in specimens.
  • Basic blue 141 also stains both mature and immature erythroblasts in b ⁇ ne marrow specimens selectively and distinctively, thereby permitting precise quantitation of erythropoiesis in bone marrow interpretation and in the dis orders of erythropoiesis, illustratively anemia.
  • Basic blue 141 also facilitates identification of early erythroblasts (proerythroblasts) which can sometimes be difficult to identify using traditional panoptic stains; medically, identification of proerythroblasts may be important in the diagnosis of various anemias and leukemias.
  • Basic blue 141 requires only one dyestuff, thereby circumventing problems involved in dye mixtures and differing lots of the same dyes.
  • Basic blue 141 does not require addition of a synthetic substrate, as in the peroxidase or specific esterase reactions. 3. Basic blue 141 does not require prior fixation. It is functional on dried but non-fixed preparations, and on methanol-fixed and FAA-fixed preparations. It is not effective as a supravital stain for neutrophils alone. 4. Basic blue 141 can be adapted as a "spot" technique, utilizing a rapid color change to red-violet as the reaction product or end point to identify the presence of neutrophils in the specimen. This is accomplished by use of an inert but integral porous matrix impregnated with the dyestuff to produce a one use, throw-away test unit.
  • This impregnated test unit wet or dry, can be applied to a suspected specimen or dipped into a specimen in an aqueous environment, allowing leukocytes to adhere to it and interact with the dyestuff to produce a highly and characteristially colored reaction product when neutrophils are prevalent.
  • Illustrative matrix material for surface coating or impregnation to produce a carrier for the basic blue 141 dye suitable for "spot" identification technique include as illustrative, but not limiting, shaped disks or rods of porous paper (filter paper) or water-wettable porous plastic forms suitable for (A) first wetting with a small retained amount of the dye and at a later time, if desired, re-wetting with the human biopsy specimen in a suitable (aqueous) environment for color change and neutrophil identification.
  • a surprising further specialized use of basic blue 141 is the utility of the dyestuff in the identification of subclasses of lymphocytes including B cells, T helper cells, T suppressor cells and Natural Killer (NK) cells with remarkable individual differentiation capability.
  • a presently developed technique found useful is to fix coverslip films of peripheral blood or bone marrow or alternatively blood smears in FAA fixative (95% ethanol - 90 ml; glacial acetic acid - 5 ml; 37% formalin solution - 5 ml). Time - 5 minutes. Wash the films in distilled water.
  • the so prepared slide is flooded with the dye solution in distilled water made by adjusting a 1% solution of the basic blue 141 dye to a pH of 5.2 to 5.5 by use of the Trizmal (Tris maleate) pH 7.6 buffer concentrate, 200 mM/ liter.
  • Trizmal Tris maleate
  • pH 7.6 buffer concentrate 200 mM/ liter.
  • a golden sheen forms on the surface of the aqueous dye solution and a color change from a blue to a blue violet is observed. Stain for about five minutes.
  • Excess dye is removed by washing the slide with distilled water; blot dry on bibulous paper. Mount the coverslip with immersion oil for use under conventional light microscopy.
  • test cell populations were originally identified by means of the following antibodies: B cells by the F(ab)2 antibodies, T helper cells by the OKT4 antibody, T suppressor cells by the OKT8 antibody and Natural Killer (NK) cells by the leu 7 antibody.
  • B cells by the F(ab)2 antibodies
  • T helper cells by the OKT4 antibody
  • T suppressor cells by the OKT8 antibody
  • Natural Killer (NK) cells by the leu 7 antibody.
  • B cells are identified by their pale green nucleus, colorless to faint blue cytoplasm and absence of granules.
  • T helper cells are distinguished by their deep blue green nucleus, very dark blue scant cytoplasm, absence of granules, but dark blue "speckles" in the nucleus.
  • T suppressor cells have a blue-green nucleus and a deep blue scant cytoplasm containing a plurality (to about 6) of tiny red granules in the cytoplasm.
  • Killer (NK) cells are larger than all of the foregoing cells and are characterized further by a very pale green nucleus and a colorless to faint blue abundant cytoplasm containing many large red granules.
  • the T suppressor and T helper cells are clearly smaller than the B-cells and the NK cells.
  • the biopsy specimen need not be freshly secured, may be dried out at toom temperatures and still provide viable and characteristic color distinctions. 2.
  • the procedure is unusually rapid, compared to the more tedious immunoiogical methods that may take substantial specimen preparation.
  • lymphocytes B, T helper, T suppressor and Natural Killer.
  • Color differentiation and enumeration can be utilized in a differential cell count, since all of the other formed elements of the blood can also be identified. In complex iramunological techniques, usually only one or two cells types can be identified.
  • the method can be used for the rapid diagnosis of the immunologic subtype of acute lymphoblastic leukemia or chronic lymphocytic leukemia, prior to the institution of specific therapy. In the case of the T cell malignancies, identification of this particular subtype may have important prognostic implications.
  • lymph node imprints particularly in the distinction between reactive hyperplasia in which T and B cells may be found in conjunction with each other, and malignant lymphoma in which all of the cells are either B or T cell type.
  • identification of these cells with a simple staining technique provides many advantages, including immediacy and inexpensiveness, compared to more complex immunologic methods presently known.
  • a 52 year old man was evaluated for weakness and fatigue. He was found to have an enlarged spleen. Laboratory tests revealed hemoglobin 7 grams %, hematocrit 22%, white blood cell count 4200/mm 3 with normal differential, and platelets 143,000/mm 3 .
  • a bone marrow specimen was [obtained. A part was stained with Wright's stain. Simultaneously, a methanolfixed coverslip containing a part of the bone marrow specimen was stained with basic blue 141. Strikingly increased numbers of mature and immature erythroblasts, along with neutrophils, were stained differentially and selectively by this dyestuff, permitting their quantitation with ease.
  • Enlarged lymph nodes could be palpated in the cervical and axillary areas, and the spleen was markedly enlarged.
  • Laboratory values revealed hemoglobin 10 gm%, white blood count 230,000/mm 3 , and platelet count 87,000/mm 3 .
  • On Wright's stain of the peripheral blood nearly all of the cells were intermediate sized lymphocytes. Immunologically using immunoperoxidase techniques, the lymphocytes were B cells. On a separate slide stained with basic blue 141, all of the lymphocytes had pale green nuclei, and cytoplasm that was virtually unstained or faint blue, characteristic of B lymphocytes. The diagnosis was chronic lymphocytic leukemia.
  • lymphoblasts A 3 year old black female child was admitted to the hospital because of irritability and intermittent fever. On physical examination, blood pressure 100/82, pulse 120/ minute, and temperature 39.5oC. Enlarged axillary lymph nodes and enlarged liver and spleen were noted. Laboratory values disclosed hemoglobin 7 gm%, white blood count 54,000/ mm 3 , and platelet count 32,000/mm 3 . On Wright's stained smear of the peripheral blood, most of the cells were lymphoblasts. Cytochemically, these lymphoblasts contained focal unipolar activity of acid phosphates, a marker for T cells. Immunologically, using monoclonal antibodies, the leukemic lymphoblasts were T cells.
  • the lymphoblasts showed dark, blue-green nuclear staining and deep blue cytoplasmic staining, characteristially found in identification of T helper cells with this dye.
  • the diagnosis was acute lymphoblastic leukemia, T cell convoluted type.
  • Accompanying Plate II details black and white sketches of the enlarged cells, pointing out verbally color descriptors which are guides to the missing color reproductions. Size relationships and color descriptors are useful, but not mathematically precise.

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Abstract

La différentiation, l'identification et l'énumération de trois types de cellules de sang humain avec le colorant bleu basique 141. Dans un type, seuls les neutrophiles mûrs parmi tous les leucocytes se teintent d'un rouge-violet vif, alors que les leucocytes immatures de tous les types se teintent de bleu pâle. Dans un deuxième type, les érythroblastes tant mûrs qu'immatures se distinguent du premier type, les uns par rapport aux autres, et de toutes les autres cellules connues susceptibles de se rencontrer dans des échantillons de biopsie humaine par un spectre différent de couleur, c'est-à-dire, une couleur cuivrée distinctive. Dans un troisième type, les diverses sous-populations de lymphocytes (cellules B, cellules T helper, cellules T de suppression et cellules tueur naturelles) se dintinguent les unes des autres par des différences caractéristiques de grandeur, de teinte et de chromaticité.
PCT/EP1985/000379 1984-08-09 1985-07-27 Differenciation, identification et enumeration de neutrophiles, d'erythroblastes et de sous-populations de lymphocytes avec du bleu basique 141 Ceased WO1986001293A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0499693A3 (en) * 1991-02-22 1993-02-03 Toa Medical Electronics Co., Ltd. Method of differentiating erythroblasts from other cells by flow cytometry
GB2355790A (en) * 1999-10-27 2001-05-02 Kay Cottington Detecting cancer
CN114222907A (zh) * 2019-08-06 2022-03-22 贝克曼库尔特有限公司 检测和报道中性粒细胞亚群

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4400370A (en) * 1980-03-12 1983-08-23 Lawrence Kass Metachromatic dye sorption means for differential determination of leukocytes
GB2116712A (en) * 1982-03-09 1983-09-28 Lawrence Kass Metachromatic dye sorption and fluorescent light emissive means for differential determination of developmental stages of neutrophilic granulocytic cells and other leukocytes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4400370A (en) * 1980-03-12 1983-08-23 Lawrence Kass Metachromatic dye sorption means for differential determination of leukocytes
GB2116712A (en) * 1982-03-09 1983-09-28 Lawrence Kass Metachromatic dye sorption and fluorescent light emissive means for differential determination of developmental stages of neutrophilic granulocytic cells and other leukocytes

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0499693A3 (en) * 1991-02-22 1993-02-03 Toa Medical Electronics Co., Ltd. Method of differentiating erythroblasts from other cells by flow cytometry
US5298426A (en) * 1991-02-22 1994-03-29 Toa Medical Electronics Co., Ltd. Method of differentiating erythroblasts from other cells by flow cytometry
GB2355790A (en) * 1999-10-27 2001-05-02 Kay Cottington Detecting cancer
CN114222907A (zh) * 2019-08-06 2022-03-22 贝克曼库尔特有限公司 检测和报道中性粒细胞亚群

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