DYESTUFF COMPOSITION FOR HISTOLOGICAL EXAMINATIONS

Abstract: New dyestuff composition for histological examinations, which comprises (1) a product formed by the interaction of one molar proportion of one or more thionine dyestuffs selected from thionine (Colour Index 52,000), toluidine blue (Colour Index 52,040) and dimethyl thionine (Azur A) with at least 0.2 molar proportion of resorcine at elevated temperature, (2) 0.5(MT/MA) to 2(MT/MA) moles of a dyestuff "A", calculated for one mole of the thionine dyestuff interacted with resorcine, and (3) 0.5(MT/MB) to 2(MT/MB) moles of a dyestuff "B", calculated for one mole of the thionine dyestuff interacted with resorcine, wherein dyestuff "A" and dyestuff "B" are alkaline dyestuffs different in colour and pH, and both dyestuff "A" and dyestuff "B" can be mixtures of alkaline dyestuffs similar in colour and pH, MT is the molecular weight of the thionine dyestuff, MA is the molecular weight of dyestuff "A", and MB is the molecular weight of dyestuff "B", or when any of the thionine dyestuff, dyestuff "A" and dyestuff "B" is a mixture of more substances, MT, MA and MB represent the weighted average of the molecular weights of the components of the thionine dyestuff, dyestuff "A" and dyestuff "B", respectively. The new dyestuff compositions according to the invention enable one to perform a wide variety of histological examinations much more quickly, simply and safely than before, and can also be applied to detect histological changes which could not be detected before.

Description: ΗΪSS FΕ COMPOSITION FOE HISTOLOGICAL EXAMINATIO The invention relates to a new dyestuff compositi for histological examinations, Histological examinations play an essential role the investigation of pharmaceutically active substances, in biological scientific studies as well as in the therap A reliable histopathological diagnosis is an indispensabl precondition of the successful treatment of certain disea and disorders, of which the early recognition of cancerou tissues is of primary importance, Histological examination methods should give relia results easy to evaluate. Moreover, the examination metho should be quick, simple and easy to perform, without requ ing specific facilities and training. The histological staining methods known so far do not fulfil these requirements in all respects. The majori of the known staining methods is difficult to perform, time consuming and requires specific attention, and the information supplied is frequently ambiguous. As an example, haematoxylin-eosin test, the most widespread routine method for tissue staining, requires about 90-120 minutes over the freezing and fixing of the section, and the resulting histological picture can be evaluated un¬ ambiguously only in the case of striking cytological dis- OMP orders. Otherwise even an approximate diagnosis, involv¬ ing the risk of serious errors, can be given only after a professional training of several years, and one should frequently rely on standard reference prβparates. Now it has been found, unexpectedly, that the new dyestuff compositions , according to the invention enable one to perform a wide variety of histological examinations much more quickly, simply and safely than before, and the resulting histological picture enables one to set up a much more reliable diagnosis than before even without any specific professional routine or training. Using the new dyestuff compositions according to the invention, histo¬ logical changes which could not be detected so far by direct staining techniques or could . not be detected at all can be recognized easily and safely. The invention relates to a new dyestuff composition for histological examinations, which consists of (1) a product formed by the interaction of one molar proportion of one or more thionine dyestuffs select- βd from thionine (Colour Index 52,000) , toluidine blue (Colour Index 52,040) and dimethyl thionine (Azur A) with at least 0,2 molar proportion of resorcine at elevated temperature, ~~ (2) 0.5(M α} /M & ) to 2(M T /M & ) moles of a dyestuff "A", calculated for one ole of the thionine dyestuff inter¬ acted with resorcine, and 1$ O^ g j /Mg to 2(M T /M B ) moles of a dyestuff "B" , calculated for one mole of the thionine dyestuff inter¬ acted with resorcine, wherein dyestuff W A M and dyestuff "B n are alkaline dyestuff different in colour and pH, and both dyestuff "A-" and dye- CMrl stuff B M can be mixtures of alkaline dyestuffs similar in colour and pH, ™ is the molecular weight of the thionine dyestuff M. is the molecular weight of dyestuff "A w , and Ilk is the molecular weight of dyestuff "B", or when any of the thionine dyestuff, dyestuff W A" and dyestuff "B" is a mixture of more substances, l_k, " M. and Mg represen the weighted average of the molecular weights of the components of the thionine dyestuff, dye- stuff "A" and dyestuff --B-*, respectively, Dyestuffs "A" and - * B n are preferably different in dispβrsity as well, " When dyestuff - • A" or dyestuff "B" is a mixture of dyestuffs, it is preferred to apply dyestuffs with similar dispersities to form the mixture, The basic component of the dyestuff compositions according to the invention is the product formed by the interaction of a thionine dyestuff with resorcine at elevated temperature. It has been found that when one mole of a thionine dyestuff is contacted with at least 0,2 moles of resorcine for a short time at elevated temperatures (preferably at 70-120°C) , a new substance, different in staining characteristics from the starting thionine dye- stuff, is obtained. It has also been observed that when a thionine .dyestuff is admixed with resorcine without heat- ing (e,g, at room temperature), the staining character¬ istics of the thionine dyestuff do not change. On this basis it is assumed that a chemical interaction takes place between the thionine dyestuff and resorcine at elevated temperatures; the nature of the product (uniform molecules, a mixture of " different reaction products, adduct, complex, etc) is, however, unknown. It is preferred to utilize 1-5 moles of resorcine for one mole of thionine dyestuff. Based on our preliminary examinations it is assumed that OλfF 3-4 moles of resorcine are required to obtain a-complete interaction with one mole of the thionine dyestuff.. The best results are obtained wieh 3-4 moles of resorcine are utilized for one mole of the thionine dyestuff; nevertheless the dyestuff composition according- to the invention-enables one to perform quick and reliable examinations when the thionine dyestnff is present in an excess not greater than 5 moles of thionine dyestuff for one-mole -of resorcine,. An excess.of resorcine does not. disturb-staining at all., it is not economical, however, to use resorcine in excess. As mentioned above, the thionine dyestuff.is contacted with resorcine at elevated temperatures. The interactio.n proceeds even at about 50°0, it is preferred, however, to contact these starting substances at higher temperatures, such as at 70-120°C, The interaction proceed almost instantaneously; thus e.g. when the starting sub¬ stances are contacted with each other in an aqueous solu¬ tion, it is sufficient to boil the solution for some seconds. Any inert liquid capable of dissolving the start¬ ing substances can be-applied as reaction medium. The . term "inert" means here that the liquid does not reac - with any of * the starting.substances. The most preferred reaction medium is water, Dyestuffs "A" and !'B° are alkaline background dyestuffs which facilitate the observation of the colour effect of the interaction product defined above. As men- tioned above, dyestuff "A" may be .a. single alkaline dye- stuff or a mixture of alkaline .dyestuffs similar in colour, pH, and-preferably in. dispersity.as well; the same holds for.dyestuff .-'B-?, It is, however, a fundamental require¬ ment that,dyestuff "A" (or dyestuff. mixture "A ** ) should differ from dyestuff "B" (or dyestuff mixture π B- * ) in colour, pH and preferably in dispersity, too * Dyestuffs., for which - the difference-in light absorption maxima does not ex¬ - th& difference in pH does not exceed 1,0, are regarded as. similar., .whereas dyestuffs, for which - the difference in light absorption maxima is at least - the difference in pH.is,at least 2,0, are regarded as different, From the-aspect of. the -similarity or difference . of alkaline dyestuffs, colour is the most, decisive factor. Thus e.g. when a dyestuff of warm colour (red, orange, yellow) or a mixture of such dyestuffs is utilized as dye- stuff "A", a dyestuff of cold colour (green, blue) or a mixture of such dyestuffs only can be applied as dyestuff "B M . ' „ , ,~... . . . . . . . . ..- it is preferred to - apply e,g. safranine (Colour Index. 50,240) and/ or Pyronin I (Colour Index 45 ,005) a s dyestuff ■ -* , together with β .g. Alcyan blue 8 GX (Colour • Inde - 74,240) and/ or Licht Grun (Colour Index 42,095) as dyestuff "B", It is preferred to apply - the two. types of back¬ ground dyestuffs in a molar ratio of 0 * 5(M A /Mg) -to 2(M./Mg) , wherein . M» and.. Mg are as defined above, . In gener the - best results are obtained with dyestuff compositions which - contain about M^/M. . mole of dyestuff "A-' and- M /Mg mole of dyestuff ,I B M for one mole of the thionine dyestuff interacted with resorcine , . A particularly preferred. dyes tuff composition con¬ tains 0.3 mole , of safranin and 0.3 mole of Alcyan blue for the interaction product of one mole of thionine with 4 moles of resorcine. The dyestuff compositions according to the inven¬ tion can be prepared by methods known per se, such as a) the interaction product of the.thionine dye- stuff and ' resorcine, separated in-solid form, is admixed with appropriate-amounts of dyestuffs "A" and "B", or b) appropriate amounts of dyestuffs tt A w and "B" are.dissolved, in the reaction, mixture (generally aqueous solution) obtained af e -the interaction of -the thionine dyestuff and resorcine, .and, if desired, the solvent is removed by known techniques, such as distillation, freeze drying, etc. The dyestuff compositions according to the . inven¬ tion can-be applied in histological examinations e.g. for the following purposes: .. ... . - * -normal" staining of nucleus and cytoplasm (for this pur pose the haematoxylin-βosin staining discussed above has been applied most frequently), - distinguishing of connective and smooth muscular element (so far the Mallory or Azan method has been used for this purpose) , - staining of mitochondria (this has been done so far e.g. with iron-haematoxylin), - distinguishing of serous and mucinous glands (no direct staining method has been known so far for this purpose; the indirect method is a staining with mucicarmin and . a simultaneous background staining), - detection of neurosecretory grains (this-has been per¬ formed so far.e.g. by the Gδmδri method), - distinguishing of cartilaginous tissues different in maturity (no appropriate method has been known so far for this purpose) , and - determination, of the ratio of nuclei active and passiv (about to disintegrate, just under disintegration, al¬ ready disintegrated) from the aspect of protein synthe (passive nuclei appear in large amounts in tumours, which could not be detected so far by any method). The known staining methods mentioned above are discussed in detail, in the following textbooks: Hans' Christian Burck: *•-His ologische Technik". (Georg. Thieme Verlag, Stuttgart 9 ' 1969; 2nd edition) .A.G.E. Pearse: "Ξistochemis ry, Theoretical and Applied (Little Brown and Co., Boston, 1968), It is clear from the above- that the dyestuff com¬ positions according to the inventio .have a very wide- range of application, and are-particularly suitable for the higtopathological diagnostics and examination of cancer. The invention is elucidated ip. detail by the aid of the following non-limiting Example, Example * 0 g of thionine and 1,30 g of resorcine are dis solved in 1000 ml of water at room temperature (about 25° and the solution is heated to boiling. After 3-5 seconds, of boiling the.solution is allowed to. cool.to about 25°C, then further 3«80 g of resorcine are.added, and- the solu- tion is boiled again-for 3-5 seconds. The,solution is allowed to cool to room temperature and 2,16 g of Alcyan blue 8 GX are added under stirring.. The solution is filte the volume of..the. filtrate is adjusted to.1000 ml with distilled water, and.2,5 g of safranin are dissolved in the mixture. The resulting solution is.filtered. again, and. the volume of the filtrate is adjusted. again to 1000 with distilled water. This aqueous solution contains OMPI 0.003 mole of Alcyan blue 8 GX, 0.003 mole of safranin, and an interaction product formed from 0,01 mole of thionine and 0.04 mole of resorcine. The solution can be used direct-ly for tissue staining. . ...If the dyestuff composition is to be stored for a prolonged period, it is sub ected to vacuum distilla¬ tion or freeze drying to remove. the solvent. 12.6 g of - a powdery dyestuff composition can be obtained from the above solution. - - The dyestuff composition can be utilized in histopathological examinations as follows: The fresh section, obtained β,g. in a surgical intervention, is frozen,and then stained for 10 minutes with the above-solution * The section is rinsed with tap water for at most- one minute, the surroundings of the - slide and the section are blotted up with tissue paper,. and then the section is treated-maximum twice with tert.- butanol for 20 seconds to effect dehydration. Thereafter the section is clarified twice with xylene for one minute, each, and then covered ith-Canada balm. Thus the sec¬ tion is ready. or histological evaluation within about 25-30 minutes, reparates for routine cytological-examinations can be made similarly with the difference that fixed sec- tions are used as starting substances. The various tissue constituents appear in the following colours: active nuclei: .blueish-green passive nuclei, nuclei before or after, disintegration (the number .of which is significant in tumorous tissues): e nucleoli: red con unc-igLve tissue.: blue smooth muscle: pink mucinous gland: blue serous gland: red ... mitochondria:- iolet grains cartilage: violet, purple, pink, blue or blueish green, ' .-depending on its maturity naurosacretory grains: red _O PI_

Claims: What we claim is: - 1, A dyestuff-composition for histological examina¬ tions, which comprises - _{1) a product formed by the interaction,of-one mola proportion of one-or. more, thionine dyestuffs selected from thionine, toluidine-blue and dimethyl thionine with at- least 0,2 molar proportion of resorcine at elevated temper ture, . .. - (2) 0.5( ) to 2(1^^) moles of a dyestuff "A", calculated for.one. mole- of the thionine dyestuff inter- " acted with resorcine, and . . .. . (3) 0«5(-l U B ) to-2(M g} / ] ^)-moles of a dyestuff "B", calculated for one ole of the thionine dyestuff intern " acted with resorcine, wherein dyestuff "A" and dyestuff. B" are alkaline dyestuffs different in colour and pH, and both dyestuff "A" and dye- stuff W B M can be mixtures o . alkaline dyestuffs similar in colour and pH, . .- . . . Mm is the molecular weight of the thionine -dyestuf , M^ is the molecular weight of dyestuff "A", and Mg is the molecular weight of dyestuff "B", or when any of- the thionine dyestuff, dyestuff "A" and dyestuff M B" is a.mix ure of more-substances, - η ,, IK and Mg represent the weighed average of the molecular weights of the components of the thionine dyestuff, dyestuff n A M and dyestuff "B", respectively, 2, A dyestuff composition as claimed in claim 1, which comprises as component (1) a product formed by the interaction of one-mole of a thionine dyestuff, preferably thionine, with 1 to 5 moles of resorcine at elevated temperature. OMPI 3 , A dyestuff composition, as claimed, in claim 1 or 2, which comprises as component ( 1) a product formed by the interaction of one mole of a thionine dyestuff, prefe ably thionine , with 3 to 4 moles of resorcine at elevated temperature. 4, A dyestuff composition as claimed in any of the preceding claims, which comprises as dyestuff "A" Alcyan blue 8 GX and as dyestuff "B" sa-franinβ, ' *" 5β A- dyestuff composition as claimed in claim 1 or 4, which comprises, dyestuffs. "A" and M B" in a molar ratio of 0.5(M A /lIg) to 2( . M A /Mg) , wherδin M^aώd Mg are as defin in claim 1, 6, A dyestuff composition as.claimed in any of the preceding claims, which comprises 0,3 moles of safranine and 0.3 moles of-Alcyan blue calculated for a product-for from the interaction of 1 mole of thionine with 4 moles of resorcine. CMPI

Inventors: BALOGH GYOERGY, CSABA GYOERGY, KOVACS PETER, BALOGH, GYOERGY, CSABA, GYOERGY, KOVACS, PETER
Applicants: REANAL FINOMVEGYSZERGYAR
Application year: 1982
Main classification: C09B47
IPC codes: G01N1