Patentamt 716 JEuropaischesEuropean Patent Office Publication number: 0183

Office europeen des brevets B1

EUROPEAN PATENT SPECIFICATION

N Date of publication of patent specification: 04.01.89 intci.4: G 01 N 1/30, G 01 33/52, G 01 N 33/48 Application number: 85902018.2 Date of filing: 12.04.85

i International application number: PCT/EP85/00162

I International publication number: WO 85/05180 21.11 .85 Gazette 85/25

IDENTIFICATION OF MYELOBLASTS AND OTHER IMMATURE GRANULOCYTIC CELLS.

Priority: 27.04.84 US 604741 Proprietor: Kass, Lawrence 1939 Ridge Road Hinckley Ohio 44233 (US) Date of publication of application: 11.06.86 Bulletin 86/24 Inventor: Kass, Lawrence 1939 Ridge Road Publication of the grant of the patent: Hinckley Ohio 44233 (US) 04.01.89 Bulletin 89/01 Representative: Kador & Partner Designated Contracting States: Corneliusstrasse 15 BECHDEFRGBITLINL D-8000 Munchen 5 (DE)

References cited: GB-A-2 074749 GB-A-2 095 402 GB-A-2116 712

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Note: Within nine months from the publication of the mention of the grant of the European patent, any person may of shall give notice to the European Patent Office of opposition to the European patent granted. Notice opposition It shall not be deemed to have been filed until the opposition fee has been Q. be filed in a written reasoned statement. paid. (Art. 99( 1 ) European patent convention). LJJ Courier Press, Leamington Spa, England. EP 0 183 716 B1 Description unstable and fades over a period of time. Further- more, some leukemic myeloblasts presently iden- Background of the invention tified as myeloblasts with monoclonal antibodies, Cytochemistry of blood cells began with Ehrlich do not contain -demonstrable peroxidase activity. in 1879. Using a triacid staining mixture com- 5 Over the past several decades, other tests have posed of orange G, methyl green and acid fuch- been devised as possible alternatives to the sin, Ehrlich obtained differential coloration of the myeloperoxidase reaction, because of the limita- various types of white blood cells. The names he tions of this reaction as noticed. applied to these cells such as neutrophils, First of these alternative tests was the Sudan eosinophils and basophils are in established use io black B stain. Sudan black B is known as Cl today. Their study forms the foundation of mod- solvent black 3 or Cl 26150. Applied to fixed ern hemotology. preparations of blood and bone marrow cells, Soon after Ehrlich's basic work, other types of Sudan black B stains lipids in the granules of stains were discovered in conjunction with efforts granulocytic cells at all stages of their develop- to demonstrate malarial parasites. The earliest of w ment. The stain is weakest when used in conjunc- these were the stains of Malachowski and tion with immature granulocytic cells, like Romanowsky in 1891. The so-called Romanowsky myeloblasts and promyelocytes. It is strongest in mixture of eosin, methylene blue, azure and mature granulocytic cells such neutrophils, eosinates soon supplanted Ehrlich's original eosinophils and basophils. Although useful in the triacid stain. At present, modifications of the 20 laboratory for distinctive identifications between Romanowsky stain, such as Wright's or Giemsa's leukemic myeloblasts leukemic lymphoblasts. stain, are used widely in hemotology for blood Sudan black B stain shows poor localization in cell identification. granular structures. Furthermore, there is con- Early in the history of blood cell identification, it siderable nonspecific background precipitation of became apparent that the differentiation and 25 the dyestuff in many of the contemporary identification of the early, immature or primitive methods for its use. precursor cells in blood and bone marrow created Along with the development of Sudan black B difficulties when using "Romanowsky" type as an alternative to the traditional myeloperoxid-' staining mixtures. To circumvent this problem, ase reaction, a specific esterase enzyme was cytochemical stains were developed. Cytochemis- 30 identified as unique to cells of the granulocytic try represents biochemistry based on studies at series. Using naphthol-ASD-chloroacetate as the microscopic and submicroscopic levels. Applied • substrate and a sensitized dye like Fast blue BBN to blood and bone marrow cells, cytochemistry as the indicator, the specific esterase stain dem- identifies mature cells and their primitive or onstrates granulocytic properties in immature immature precursors on the basis of a charac- 35 cells like myeloblasts in acute myeloblastic teristic property, such as a unique enzyme or leukemia or in granulocytic sarcoma (chloroma). cellular metabolite, rather than on physical This stain and method has several shortcomings. features such as size, shape or color. These include the need for an exogenous sub- Among the earliest cytochemical stains to be strate, use of a sensitized unstable dye coupler, applied to the problem of blood and bone marrow 40 imprecise localization of the reaction product in cell identification was the peroxidase stain. Used granules, need for a nuclear counterstain, pro- initially as a measure of the oxidizing ferment in longed incubation period to perform the test, and vegetables, the peroxidase staining method was considerable background precipitation of the applied to identification of blood cells about 75 coupler, making it difficult to distinguish between years ago. Soon, it became apparent that some 45 artifact and reaction product. cells contained peroxidase activity (granulocytes) As a result of the shortcomings of the specific and other cells did not (lymphocytes). Not long esterase reaction, other stains have been afterwards, the peroxidase stain was applied to developed as alternatives. Used as direct stains the study of immature or primitive cells in acute applied to fixed preparations of blood and bone leukemia. For the first time it became possible to so marrow cells, these later stains were simpler in distinguish immature or primitive granulocytic application than the traditional stains and have cells, called myeloblasts, from immature lym- produced comparable results. phoid cells, or lymphoblasts. Complexities associated with the specific ester- This distinction could be achieved on the basis ase reaction tests led to the discovery that of the detection of myeloperoxidase activity in 55 chlorozol black E (direct black 38, Cl 30235) in myeloblasts but not in lymphoblasts. which both primary granules or lysosomes, and In the customary practice of cytochemical stain- secondary granules or specific granules have ing of myeloperoxidase requires the addition of a been distinguished and demonstrated in neut- color forming compound when oxidized, an rophilic granlocytic cells on the basis of identifi- exogenous chromogen, commonly benzidine or so able differences. o-tolidine, coupled with an addition of an oxidiz- Using Saturn blue; Cl 42045, also known as acid ing agent, illustratively hydrogen peroxide. blue 1; similar differences in primary and In the case of benzidine, for example, it is a secondary granules have been noted. suspected carcinogen and is of potential hazard to Using Niagara sky blue 6B; Cl 24410, also the user. The reaction product is unfortunately 65 known as direct blue 1; identifiably differential EP 0183 716 B1 coloration of primary and secondary granules Subsequently the fixed specimen slide pre- provided an advance in the art. paration is rinsed in distilled water. An aqueous With the acid dye known as sulfonaphthyl red, solution of basic blue 93 is applied to the rinsed an acid dye not identified by Colour Index surface of the specimen and stained for about number, both primary and secondary granules 5 three minutes, washed again with distilled water are stained red. to remove excess dye color and mounted with So far as presently known, no dyestuff has Permount (or equivalent on cleaned glass slides heretofor been suggested or reported which for normal light microscope examination. selectively and preferentially differentially stains Upon examination of normal peripheral blood only primary granules or lysosomes in dried and/ 10 samples, only a few tiny black granules have been or fixed cells of the neutrophilic granulocytic observed in normal neutrophils and bands. More series including myeloblasts, promyelocytes, granules were observed in the field with bands myeolocytes, bands and neutrophils. than were apparent with the neutrophils. Black Heretofor, all of the useful stains have been granules as were noticed of the type found in the either acid dyes or direct or solvent dyes and non 15 neutrophils were not visually apparent among the have been basic cationic dyes. None of the fore- eosinophils, basophils, lymphocytes, monocytes, going have been selective, since they stain both erythrocytes or platelets present. primary and secondary granules, producing in Normal bone marrow aspirates stained with some instances color differences. basic blue 93 revealed nuclei of all cells to be 20 stained pale purple whereas the cytoplasm of the Detailed description of the invention cells were less red, a pale lavender. The dyestuff of this invention as used in Claim 1 In cells of the neutrophilic granulocytic series, and applied in the method of Claim 2 is the sole primary granules or lysosomes were stained an basic cationic dyestuff presently known in the art intense black. Promyelocytes also exhibited large which preferentially and selectively stains prim- 25 numbers of granules which were stained black. In ary granules or lysosomes in the cells of the study of the myelocytes fewer numbers of granu- neutrophilic granulocytic series. les were noted as compared with promyelocytes. The remarkable basic cationic dye of this inven- Among the metamyelocytes, bands and neut- tion known generally as basic blue 93 has not rophils only a few tiny black granules were been publicly identified by chemical structure, nor 30 observed. identified in the Colour Index by number. It is The granules in basophils and eosinophils were available under the trade name "Aizen Cathilon stained from a pale lavender to a cream color. No Navy Blue RLH" from Hodagaya Chemical Com- black granules were observed. Likewise, no black pany of Japan and has the absorbance spectra granules were found among the megakaryocytes, recorded in Figure 1. 35 mast cells, plasma cells, erythroblasts and his- As a result of a comprehensive testing program tiocytes of normal bone marrow aspirates as of nearly 5,000 synthetic organic dyestuffs from above. world-wide sources, basic blue 93 has been the Examination of leukemic lymphoblasts stained only dye found that preferentially and intensely with basic blue 93 as above revealed both nuclei stains primary granules or lysosomes in cells of 40 and cytoplasm to be stained a pale purple. No the neutrophilic granulocytic series, including black granules were detected. Prominent darker myeloblasts and immature granlocytic cells purple aggregates of nuclear chromatin are including promyelocytes as illustrative. Basic blue observable in the nuclei of leukemic lymphoblasts 22 or Astiazon blue FGLN is a poor second. stained with basic blue 93. In leukemic myelob- In reduction to practice of the invention a stock 45 lasts from patients with acute myeloblastic solution of the dye, basic blue 93, is dissolved in leukemia, black granules were found, their num- distilled water. A dye concentration of 10% has bers varying in the cytoplasm of many of the been found useful and no criticality appears to be leukemic blasts. These observations were con- associated with an unbuffered solution which firmed in parallel slides where peroxidase stain shows no apparent deterioration or settling or so and Sudan black B stains also gave similar posi- evidence of change in quality over storage at tive confirmation. However, in some instances room temperature for at least one full year. The where the parallel and comparative slides in color of the aqueous solution is broadly described which Sudan black B stain and the peroxidase as a reddish-purple. stain were negative in response, basic blue 93 A preferred laboratory procedure has been to 55 staining was positive. The leukemic blasts were prepare coverslips with human biopsy speci- confirmed to be myeloblasts when checked out mens, illustratively; blood, blood and bone through use of monoclonal antibodies. marrow, imprints of bone marrow biopsy core, Leukemic blasts from patients known to suffer lymph node imprints and the like by saturation of acute promyelocytic leukemia, stained with basic the specimen in absolute methanol as the fixative 60 blue 93 as above, revealed large numbers of black for about two minutes. (Oddly, the stain is oper- stained granules. Auer rods, a marker for ative in aqueous solution when applied to dried leukemic blasts of granulocytic origin, also unfixed cells. However, there is superior and stained black. preferred localization of the reaction product Biopsy specimens from patients with acute when the stain is applied to fixed cells). 65 myelomonocytic leukemia, similarly stained, dis- EP 0 183 716 B1 closed leukemic monocytes having a few black nation, he had normal vital signs. The liver and granules in the cytoplasm confirming the spleen were enlarged. Laboratory values included granulocytic origin of the cells. Leukemic mono- hemoglobin 8.2 g%, white blood cell count cytes from a patient known to suffer from acute 135,000/mm3, and platelet count 32,000/mm3. histiomonocytic leukemia, comparatively s On Wright's stain of the peripheral blood and examined, did not reveal the presence of black bone marrow, many of the cells were leukemic stained granules. blasts with delicate nuclear chromatin pattern and Prior art stains presently known and used to basophilic cytoplasm devoid of granularity. identify cells of the neutrophilic granulocytic Using Sudan black B and myeloperoxidase series including, as illustrative, myeloperoxidase, 10 staining, a few of the leukemic blasts showed Sudan black B, specific esterase and the new activity of peroxidase and faint staining with stains, amongst which are direct black 38 and acid Sudan black B. blue 1, lack the following several important Using basic blue 93, many of the leukemic advantages in direct comparison to basic blue 93. blasts, contained black punctate granules in the These include the following: 15 cytoplasm indicating more clearly their 1. Identification of both myeloblasts and prom- granulocytic origin than was manifest in the use yelocytes is more accurate than heretofor due to of Wright's stain and Sudan black B. unusually precise localization of the dye-cell reac- Diagnosis made: Acute myeloblastic leukemia. tion product in the lysosomes of immature granulocytic cells: 20 Example 2 2. The dye-cell reaction product is jet black. It is A 38 year old black female was admitted to the easily visible in contrast to the pale lavender color hospital because of nosebleeds. On physical of the nucleus. examination, her vital signs were normal. The 3. Basic blue 93 differentially stains both nuc- patient had slightly enlarged lymph nodes in the leus and granules, thus a separate counterstain 25 neck and groin. Laboratory data included hemog- for the nucleus is unnecessary. lobin 7.1 g%, white blood cell count 83,000/mm3, 4. The subject stain is both selective and and platelet count 22,000/mm3. Evidences of dis- specific for primary granules (lysosomes). Cells/ seminated intravascular coagulation, with low including 'myeldblasts, promyelocytes and level of fibrinogen, and increased levels of fibrin myelocytes containing a preponderance of these 30 split products were observed. granules are more accurately differentiated and On Wright's stain of peripheral blood and bone identified than heretofor. marrow, many leukemic blasts were seen. Some 5. Virtually no confusing background pre- blasts contained multiple Auer rods and many cipitates occur. The problem of delineation granules. between reaction products and non-specific pre- 35 Using Sudan black B and myeloperoxidase cipitation is made minimal. stains, a few granules and Auer rods could be 6. Addition of an exogenous substrate, as in the recognized in the leukemic blasts. specific esterase reaction, for example, or addi- Using basic blue 93, many granules were seen tion of oxidizing agents, illustratively hydrogen in the leukemic blasts. Granules stained in intense peroxidase, as in the myeloperoxidase reaction, is 40 black. Multiple Auer rods were easily isolated and no longer required. identified by characteristic shape and intense 7. Dye couplers, which require pre-sensitization black color. More Auer rods and more granules before use, illustratively hexazotization of a were enumerated than with comparative use of coupler in the specific esterase reaction, are no the prior art dyestuffs above. longer needed. 45 Diagnosis made: Acute promyelocytic 8. The dye-specimen reaction product shows no leukemia. detectable fading with time (tests over a year). 9. The dye is applicable to the immediate and. Example 3 rapid diagnosis of acute leukemia where delay in A 22 year old white male admitted to the institution of treatment may be detrimental. This 50 hospital complaining of weakness and fatigue. contrasts with use of the prior art specific esterase Physical examination showed patient with normal reaction where incubation may require as long as vital signs. Enlarged lymph nodes and enlarged thirty minutes. liver and spleen were detected. Laboratory values 10. Delineation of nuclear chromatin in the included hemoglobin 6.3 grams%, white blood identification of leukemic lymphoblasts is made 55 cell count 5,600/mm3, and platelet count 12,000/ more specific and definite. mm3. The following examples are illustrative of com- Wright's stain of his peripheral blood and bone parisons in diagnostic studies of patients using marrow showed large numbers of leukemic blasts prior art dyes along with parallel comparisons with coarse appearing nuclear chromatin and using basic blue 93. In all cases fixed biopsy 60 basophilic cytoplasm devoid of granularity. specimens were compared. Using Sudan black B and myeloperoxidase stains, no granules were found to be identified in Example 1 the cytoplasm of the leukemic blasts and the A 40 year old white male was admitted to the distinctive features of nuclear chromatin could hospital with fever and chills. On physical exami- 65 not be identified. EP 0 183 716 B1 8

Using basic blue 93, no granules were seen in Granulocytic sarcoma (chloroma) was diag- the leukemic blasts. Nuclear chromatin displayed nosed. Within 4 months the patient expired with a distinctive pattern of prominent lavender purple acute myeloblastic leukemia. colored aggregates. None of the above stains In the above examples the human biopsy speci- developed a similar visible pattern of nuclear 5 mens were fixed before stainipg with basic blue that aggregates in the cytoplasm of the leukemic 93. However, it has also been determined blasts. basic blue 93 is operative when applied on or to Using specific monoclonal antibodies, the diag- dried but unfixed cells in an aqueous environment. nosis of acute lymphoblastic leukemia was con- However, use of fixed cells provides a more firmed. 10 sharply localized reaction product and the nuclear detail is improved. Oddly, the dye appears to act Example 4 both as a fixative as well as a stain. The dye is Upon hospital admittance of a black female, inoperative as a supravital stain for lysosomes. aged 34, experiencing fever and pain in the right upper quadrant, physical examination reported a is Claims temperature of 101° and tenderness to palpitation in the pain area. Laboratory reports detailed 1 . The use of a basic cationic dye known as basic homoglobin 13 gram%, white blood count 22,000/ blue 93 and which dye is further fully identified by mm3 and platelets 300,000/mm3. Ultrasound gall its known absorbance spectrum for differentiation blader examination showed multiple stones 20 and identification of normal and abnormal cells of present. the neutrophilic granulocytic series whereby the Wright's stain of peripheral blood reported reaction product of a dried and/or fixed human larger numbers of neutrophils, bands and a few biopsy specimen in an aqueous environment metamyelocytes were reported. containing at least one of the said cells in the On staining with myeloperoxidase, activity of 25 defined series is obtained with said dye. both normal the enzyme was found in all granulocytic cells, 2. A method for differentiation of particularly promyelocytes and myelocytes. and abnormal cells of the neutrophilic granulocy- Sudan black B confirmed staining of neutrophils tic series and identification of individual ones of was reported. said cells present in a dried and/or fixed human Using basic blue 93, intense black granules were 30 biopsy specimen which comprises reacting a basic found in more numerous frequency in the promy- cationic dye having the absorbance spectra of elocytes and myelocytes. A relative few granules basic blue 93 in an aqueous environment with said were observed in granulocytic bands and neut- specimen prior to analytical differential examina- rophils. The granules were, when present, more tion and identification of selected individual ones numerous and more intensely stained than with 35 of said granulocytic cells present in said specimen. the prior art stains above. Identification of imma- 3. The method of Claim 2, in which the identifi- ture leukocytes was more certain with the basic able cells are normal neutrophilic granulocytic blue 93 dyestain. Bone marrow examination cells including myeloblasts, myelocytes, promy- showed no evidence for leukemia. elocytes, metamyelocytes, bands and nuetrophils. The diagnosis of neutrophilic leukocytosis with 40 4. The method of Claim 2, in which the abnormal "shift to the left" resulted. After cholecystectomy, cells are leukemic cells of granulocytic origin the patient's blood counts returned to normal. including leukemic myeloblasts and promyelocy- tes that contain Auer rods marking their Example 5 granulocytic origin. An oriental female, 38 years, was admitted to the 45 5. The method of Claim 2, applied to leukemia hospital with' a rapidly enlarging mass in the left diagnosis which differentiates leukemic myelob- cervical area. Physical examination established lasts and leukemic promyelocytes by the black presence of 3x4 cm mass and normal vital signs. reaction product in their lysosomes from leukemic Laboratory analysis included hemoglobin 14 lymphoblasts which do not contain the said black grams%, white blood cell count 8,500/mm3 with so reaction product. normal differential and platelet count 205,000/ 6. The method of Claim 5, wherein the leukemic mm3. The biopsied mass proved to be an abnor- cells of granulocytic origin contain stained Auer mal lymph node. A plurality of lymph node rods in the black reaction product. imprints were made. Wright's stain indicated the presence of a large 55 Patentanspriiche number of primitive appearing cells. Test with stains for myeloperoxidase, specific 1. Anwendung eines basischen kationischen esterase and Sudan black B established a few of Farbstoffes, der als basisches Blau 93 bekannt ist the cells to contain a black reaction product und daruberhinaus durch sein bekanntes Absorp- suggesting their granulocytic origin. 60 tionsspektrum vollstandig identifiziert ist, zur Dif- Further checking with basic blue 93 established ferenzierung und Identifizierung normaler und many of the primitive cells contained numerous anormaler Zellen on neutrophilen Granulozytrei- black, granular reaction products more prominent hen, wodurch das Reaktions-produkt einer than observed in the prior tests above. Identifica- getrockneten und/oder fixierten Human-Biopsie- tions of immature leukocytes were more precise. 65 probe in einer wassrigen Umgebung, die zumin- EP 0 183 716 B1 10 dest eine dieser Zellen in diesen definierten Rei- par son spectre d'absorbance connu — pour la hen enthalt, mit diesem Farbstoff erhalten wird. differenciation et I'identification de cellules nor- 2. Verfahren zur Differenzierung sowohl norma- males et anormales des la serie granulocytaire ler als auch anormaler Zellen von neutrophilen neutrophile, par laquelle le produit de reaction Granulozytreihen und zur Identifizierung von ein- 5 d'un echantillon de biopsie humaine seche et/ou zelnen dieser Zellen, die in einer getrockneten fixe dans un environnement aqueux contenant au und/oder fixierten Human-Biopsieprobe vorhan- moins une desdites cellules dans la serie definie, den sind, welches die Reaktion eines basischen est obtenu avec le dit colorant. kationischen Farbstoffes mit dem Absorptions- 2. Methode pour la differenciation a la fois des spektrun von basisch Blau 93 in wassriger Umge- w cellules normales et anormales des series granu- bung mit dieser Probe vor der analytischen Differ- locytaire neutrophile et identification de celles entialpriifung und Identifizierung von ausgewahl- individuelles desdites cellules presentes dans un ten einzelnen dieser in der Probe vorhandenen echantillon de biopsie humaine seche et/ou fixe Granulozytellen umfaSt. qui comprend la reaction d'un colorant cationique 3. Verfahren nach Anspruch 2, worin die identi- w basique ayant le spectre d'absorbance du bleu 93 fizierbaren Zellen normale neutrophile Granulo- basique dans un environnement aqueux avec zytzellen sind, die Myeloblasten, Myelozyten, Pro- ledit echantillon prealablement a I'examen diffe- myelozyten, Metamyelozyten, Ligamente und rentiel analytique et I'identification de celles indi- Nuetrophile einschlieSen. viduelles selectionnees desdites cellules granulo- 4. Verfahren nach Anspruch 2, worin die anor- 20 cytaires presentent dans ledit echantillon. malen Zellen Leukamiezellen mit granulozyti- 3. Methode selon la revendication 2, dans schem Ursprung sind, die Leukamiemyeloblasten laquelle les cellules identifiables sont des cellules und -promyelozyten einschlielSen, die Auer-Stab- granulocytaires neutrophiles normales incluant chen enthalten, die ihren granulozytischen les myeloblastes, myelocytes, promyelocytes, Ursprung markieren. 25 metamyelocytes, bandes, et neutrophiles. 5. Verfahren nach Anspruch 2, das zur Leuka- 4. Methode selon la revendication 2, dans miediagnose angewendet wird, welches Leuka- laquelle les cellules anormales sont des cellules miemyeloblasten und Leukamiepromyelozyten leucemiques d'origine granulocytaire incluant les durch das schwarze Reaktionsprodukt in ihren myeloblastes et les promyelocytes leucemiques Lysosomen aus Leukamielymphoblasten diffe- 30 qui contiennent des batonnets d'Auer marquant renziert, die dieses schwarze Reaktionsprodukt leur origine granulocytaire. nicht enthalten. 5. Methode selon la revendication 2, appliquee 6. Verfahren nach Anspruch 5, worin die Leuka- au diagnostic de la leucemie qui difference les miezellen mit granulozytischem Ursprung im myeloblasts leucemiques et les promyelocytes schwarzen Reaktionsprodukt gefarbte Auer-Stab- 35 leucemiques par le produit noir de reaction dans chen enthalten. leur lysosomes a partir de lymphoblastes leuce- miques qui ne contiennent pas ledit produit noir Revendications de reaction. 6. Methode selon la revendication 5, dans 1. Utilisation d'un colorant cationique, basique, 40 laquelle les cellules leucemiques d'origine granu- connu comme etant le bleu 93 basique — et lequel locytaire contiennent des batonnets d'Auer le colorant est en outre completement identifie colores dans le produit noir de reaction.

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FIGURE I

VIS 350 400 450 500 550 600 650 700 750

WAVELENGTH IN nm

BASIC BLUE 93