Stabilized stain composition

Formulations of a cationic dye component (e.g., Azure A-Azure B-Azure C mixtures), an anionic dye component (e.g., Eosin Y) and an alcohol solvent (e.g., methanol or methanol-glycerol mixtures) are stabilized by an amino acid or its acid addition salt (e.g. lysine hydrochloride or glycine or a mixture of these).

BACKGROUND OF THE INVENTION 
The present invention relates to compositions for staining biological 
tissue of the type comprising a cationic dye component, an anionic dye 
component, a stabilizer and an alcohol-containing solvent; and especially 
to such compositions of the Romanowsky type. 
Romanowsky-type stains, and especially Wright stains, provided in an 
alcohol solution, are commonly used to stain biological tissue such as 
blood smears, malarial parasites and bone marrow. Unstabilized 
compositions are prone to reactions during storage that produce an 
undesired precipitate and loss of staining ability. A number of proposals 
have been made regarding the mechanism of this reaction and compositions 
have been prepared which are stabilized in various ways to counteract 
these problems. Some of the proposals have involved pH control (Gilliland 
et. al. Stain Technology Vol. 54, No. 3, pp. 141-150 (1979)), introduction 
of the anionic dye component (especially Eosin Y) in the free acid form 
rather than the alkali metal salt form (U.S. Pat. No. 4,392,864 to 
Helfrich et al (1983)) or addition of stabilizers (U.S. Pat. Nos. 
4,290,769 (1981) and 4,382,075 (1983), each to Liao et al). 
Among the stabilizers proposed have been ammonium halides and primary, 
secondary or tertiary alkylamine hydrohalides having alkyl chains of 1-6 
carbons. Especially mentioned have been diethylamine hydrochloride and 
dimethylamine hydrochloride; both of which are secondary amines of 
petrochemical origin. 
BRIEF DESCRIPTION OF THE INVENTION 
It has been found that amino acids and their acid addition salts, and 
especially alpha amino acids and their acid addition salts, are effective 
stabilizers of alcoholic solutions of Romanowsky type stains. Accordingly, 
the present invention provides a stabilized stain composition comprising: 
(a) a cationic dye component, 
(b) an anionic dye component, 
(c) an alcohol solvent, and 
(d) a stabilizer selected from the group consisting of amino acids, 
substituted amino acids and acid addition salts of amino acids. The 
prevent invention further provides a method of staining biological tissue 
which comprises applying to the tissue a stored composition comprising 
above elements (a), (b), (c), and (d). 
DETAILED DESCRIPTION OF THE INVENTION 
The present composition contains as essential ingredients two dye 
components, a solvent and a stabilizer. 
A first component in the composition of the present invention is a cationic 
dye component of the type used in various classes of biological stains. 
Preferred are the various thiazine components caused by the oxidative 
demethylation of Methylene Blue: 
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Methylene Blue +3/2O.sub.2 -- CO.sub.2 -- H.sub.2 O 
.dwnarw. 
Azure B +3/2O.sub. 2 -- CO.sub.2 -- H.sub.2 O 
.dwnarw. 
Azure A +3/2O.sub. 2 -- CO.sub.2 -- H.sub.2 O 
.dwnarw. 
Azure C +3/2O.sub. 2 -- CO.sub.2 -- H.sub.2 O 
.dwnarw. 
Thionin 
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While the above reactions show the primary products wherein methyl is 
oxidized completely to CO.sub.2 in each instance, partial oxidation to 
CH.sub.2 O, CH.sub.3 OH and HCOOH may also occur. 
One typically uses either the crude mixture of all five components or 
purified Azure A; nevertheless other combinations or partially purified 
cuts may be used. Furthermore, other cationic dye components of the type 
found in biological stains may be used: oxazines Methylene Green, 
Methylene Violet, Toluidine Blue O and the like. 
The anionic dye component of the composition is generally a fluroescein 
derivative or fluroescein itself; with the preferred anionic dye component 
being tetrabromo-fluorescein (commonly called Eosin Y). Such cationic dye 
components may be used in acid or salt forms (such as the common sodium 
salt form of Eosin Y). 
The concentrations and proportions of the dye components are not 
independently critical, but it is generally desirable to approximate the 
stoichiometry that will be followed on staining (e.g. 2:1 mole ratio of 
thiazine to eosin in most Romanowsky-type stain compositions). Departures 
from stoichiometry by either component of up to 50% in molar excess are 
contemplated, however. Wright Stain, for example, is an approximately 
1:1:1 mixture (by moles) of Methylene Blue, Azure B and Eosin Y. 
Solubility limits are normally approached as to the total concentration of 
anionic and cationic dye components. 
The solvent used is an alcohol or alcohol mixture, generally of six carbons 
or less. For obtaining rapid evaporation, the major alcohol component is 
preferably methanol, ethanol, or isopropanol, and is most preferably 
methanol. Glycerol or other diols or triols or sorbitols may also be 
present to improve stability somewhat. For some applications, however, the 
concentration of polyols and especially glycerol is preferably kept below 
5% (w/v) and more preferably is kept in the 0.5-1.5% range. For other 
applications (e.g., Wright-Giesma stains), much higher levels (e.g., 50%) 
of glycerol or the like are used. 
The stabilizer used in the present invention is an amino acid or acid 
addition salt thereof, used in an effective amount to stabilize the stain 
composition against undesired precipitate forming during storage, and 
especially against the progressive formation of such precipitates over a 
period of a few weeks or less. A preferred class of such stabilizers are 
the alpha amino acids and acid additions salts thereof because of their 
relatively innocuous character, being naturally occuring substances in 
biological materials. Among the amino acids, either D or L forms (or even 
other stereoisomers such as the meso form) may be used, or mixtures such 
as racemic mixtures may be used. L-amino acids are preferred. All classes 
of amino acids (cationic such as lysine or histidine, neutral such as 
glycine or anionic such as aspartic or glutamic acid) may be used; the 
aromatic amino acids tryptophan, phenylalanine and tyrosine, may also be 
used. Most preferred are lysine, glycine, lysine acid addition salts 
(especially the monoacid addition salts) and glycine acid addition salts. 
Among the acid addition salts, hydrohalides are preferred and 
hydrochlorides are more preferred. Esters of amino acids and their acid 
addition salts may also be used. 
While not wishing to be bound to the mechanism by which the present 
stabilizers work, it is postulated that a trace of aldehyde may form in 
stain compositions by an oxidation (e.g., of methanol to formaldehyde) and 
that such trace aldehydes may react with the phenolic and amino dyes to 
form condensates which precipitate. Amino acids can scavenge the trace 
aldehydes (forming CH.sub.2 .dbd.N--CHR--COOH from formaldehyde and alpha 
amino acid), thus preventing the precipitating condensates. 
Stated differently, a preferred class of stain compositions comprise: 
(a) a mixture of Azures A, B, and C, 
(b) Eosin Y, 
(c) methanol, and 
(d) an alpha amino acid or acid addition salt of an alpha amino acid (which 
is more preferably lysine, a lysine acid addition salt, glycine or a 
glycine acid addition salt). 
Such preferred class of stain composition may also contain glycerol. In 
such composition, components (a) and (b) (in approximately stoichiometric 
ratios) typically comprise 0.1-1.0% (w/v) of the total composition, 
component (d) typically comprises 0.0-2.0% (w/v) of the total composition 
and methanol the balance (except for up to 5% glycerol which may be 
present in Wright Stain or about 50% glycerol which may be used in 
Wright-Geisma stain).

EXAMPLES 1-5 
Compositions were prepared by mixing the ingredients shown in Table 1. All 
units are grams, except for methanol and glycerol, which are expressed in 
milliliters. 
TABLE 1 
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Formulation: 
1 2 3 C4 5 
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Ingredients: 
Azure A 3 3 3 3 -- 
Eosin Y 3 3 3 3 -- 
(Na.sub.2) 
Wright Stain 
-- -- -- -- 3 
Glycerin 2 -- 2 -- 2 
Lysine - HCl 
4 4 -- -- -- 
Diethylamine 
-- -- -- 4 -- 
HCl 
Methanol 2000 2000 2000 2000 2000 
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Two weeks after formulation, the five compositions were tested on smears of 
human blood. Ratings (based upon a value of 4.0 for a fresh commercial 
product) were 3.8 for formulations 2 and 3, 3.7 for formulation 1, 1.5 for 
formulation 5 and 1.0 for control formulation 4. These results suggest 
that either lysine HCl or glycerol (but not both together if glycerol is 
2%) are highly effective in this type of formulation; and that 
diethylamine hydrochloride was least effective. 
EXAMPLE 6 AND 7 
Formulations were then prepared with 1.5 g of Azure A (80% purity), 1.5 g 
of Eosin Y (85% purity of the disodium salt), 5 g of glycerol, one liter 
of methanol and 2 g of either lysine hydrochloride (in Example 6) of 2 g 
of glycine (in Example 7). Portions of each formulation were stored at 
25.degree. C. and at 37.degree. C. and were taken periodically and assayed 
for Eosin Y (absorption at 525 mm), for Azure A (absorption at 640 mm) and 
staining score. 
The lysine-containing formulation (Example 6) showed excellent (over 90%) 
retention of Eosin Y content up to 150 days at 37.degree. C. (somewhat 
less at 25.degree. C.), and staining scores that declined only very 
slowly, being 90% of ideal and 90% of the initial value after 150 days at 
25.degree. C. Larger scale batches performed somewhat similarly. 
The glycine-containing formulation of Example 7 also showed excellent 
retention of values for Eosin Y and Azure A based upon absorbance. The 
staining scores were also quite good through 270 days storage, except that 
performance began deteriorating badly after 2 months exposure to 2.degree. 
C. When, however, lysine hydrochloride and glycine were present together 
in the formulation, staining performance was good even at 2.degree. C. and 
-10.degree. C. 
Compositions have also been prepared containing lysine hydrochloride and 
glycine and glycerol. All performed well when stored at 25.degree. C., and 
the glycine-containing formulations performed well when stored and/or used 
at lower temperatures (typically 2.degree. C.).