FIXATIVE AND STAINING SOLUTIONS
The formulations, systems, and methods disclosed herein permit automated preparation of specimens (e.g., biological specimens) for examination. The disclosed formulations, systems, and methods provide fast, efficient, and highly uniform specimen processing using minimal quantities of fluids. The methods include at least a fixing phase for fixing a specimen to a substrate such as a microscope slide, a staining phase for staining the specimen, and a rinsing phase for rinsing the specimen. One or more of the fixing, staining, and rinsing phases include one or more agitation phases for distributing reagents evenly and uniformly across the specimen. The systems can be implemented as a standalone device or as a component in a larger system for preparing and examining specimens.
1 . A cytological staining solution, comprising
Azure B;
methylene blue;
a buffering agent;
a surfactant,
sodium chloride, and
water.
2 . The cytological staining solution of claim 1 , further comprising an antimicrobial agent.
3 . The cytological staining solution of claim 2 , wherein the solution comprises from about 0.2 to about 50 ppm of the antimicrobial agent.
4 . The cytological staining solution of claim 2 , wherein the antimicrobial agent is selected from the group consisting of benzalkonium chloride, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, ProClin®, azides, merthiolates, antibiotics, and any combination thereof.
5 . The cytological staining solution of claim 2 , wherein the antimicrobial agent comprises 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one. 41. The cytological staining solution of claim 40 , wherein the antimicrobial agent is ProClin® 300.
6 . The cytological staining solution of claim 1 , further comprising acetic acid. 43. The cytological staining solution of claim 36 , wherein the solution comprises from about 0.25 to about one g/L azure B.
7 . The cytological staining solution of claim 1 , wherein the solution comprises from about 0.25 to about one g/L methylene blue.
8 . The cytological staining solution of claim 1 , wherein the solution has a pH of from 5 to 8 and a buffering agent concentration of from about 5 mM to about 250 mM.
9 . The cytological staining solution of claim 1 , wherein the buffering agent comprises bis-tris buffer, phosphate, HEPES, MES, Tris, and any combination thereof.
10 . The cytological staining solution of claim 9 , wherein the solution has a pH of from 6.8 to 7.2 and a bis-tris buffer concentration of from about 25 mM to about 100 mM.
11 . The cytological staining solution of claim 1 , wherein the solution comprises from about 0.05 to about 0.5% by volume of the surfactant.
12 . The cytological staining solution of claim 11 , wherein the solution comprises from about 0.05% to about 0.3% by volume of the surfactant.
13 . The cytological staining solution of claim 1 , wherein the surfactant is selected from the group consisting of non-ionic, cationic, anionic, and zwitterionic surfactants.
14 . The cytological staining solution of claim 13 , wherein the non-ionic surfactant is polysorbate 20.
15 . The cytological staining solution of claim 14 , wherein the solution comprises from about 0.5 mL/L to about two mL/L of polysorbate 20.
16 . The cytological staining solution of claim 1 , wherein the solution comprises from about one to about 20 g/L sodium chloride.
17 . The cytological staining solution of claim 1 , wherein a 1:1000 dilution of the solution in water has a UV absorbance of from about 0.1 to about one at a peak wavelength of from about 640 to about 660 nm.
18 . The cytological staining solution of claim 1 , wherein the surfactant is non-ionic.
19 . A cytological staining solution, comprising
about 0.25 g/L to about 2.5 g/L Azure B;
about 0.25 g/L to about 2.5 g/L methylene blue;
about 5 mM to about 250 mM bis-tris buffer;
about 0.5 mL/L to about 2.0 mL/L polysorbate 20,
about 1.0 g/L to about 20 g/L sodium chloride, and
about 0.2 ppm to about 50 ppm ProClin 300<(R)>;
acetic acid; and
water,
wherein the solution has a pH of from 6.8 to 7.2.
20 . A rinse solution for an automated specimen preparation apparatus, comprising
polyethylene glycol;
a buffering agent;
a surfactant;
methanol; and
water.
21 . The rinse solution of claim 20 , further comprising an antimicrobial agent.
22 . The rinse solution of claim 21 , wherein the solution comprises from about 0.2 to about 50 ppm of the antimicrobial agent.
23 . The rinse solution of claim 21 , wherein the antimicrobial agent is selected from the group consisting of benzalkonium chloride, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, ProClin®, azides, merthiolates, antibiotics, and any combination thereof.
24 . The rinse solution of claim 21 , wherein the antimicrobial agent comprises 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one.
25 . The rinse solution of claim 24 , wherein the antimicrobial agent is ProClin® 300.
26 . The rinse solution of claim 20 , wherein the solution comprises from about 0.2 to about two g/L polyethylene glycol.
27 . The rinse solution of claim 20 , wherein the solution has a pH of from 5 to 8 and a buffering agent concentration of from about one mM to about 50 mM.
28 . The rinse solution of claim 20 , wherein the buffering agent comprises bis-tris buffer, phosphate, HEPES, MES, Tris, and any combination thereof.
29 . The rinse solution of claim 20 , wherein the solution comprises from about 0.01 to about 0.1% by volume of the surfactant.
30 . The rinse solution of claim 29 , wherein the solution comprises from about 0.0501% to about 0.306% by volume of the surfactant.
31 . The rinse solution of claim 20 , wherein the surfactant is selected from the group consisting of non-ionic, cationic, anionic, and zwitterionic surfactants.
32 . The rinse solution of claim 31 , wherein the non-ionic surfactant is polysorbate 20.
33 . The rinse solution of claim 32 , wherein the solution comprises from about 0.1 mL/L to about 0.4 mL/L of polysorbate 20.
34 . The rinse solution of claim 20 , wherein the solution comprises from about 9 to about 11 mL/L methanol.
35 . The rinse solution of claim 20 , wherein the surfactant is non-ionic.
36 . A rinse solution for an automated specimen preparation apparatus, comprising:
about 0.2 g/L to about 10 g/L polyethylene glycol;
about 1 mM to about 250 mM HEPES buffer;
about 0.10 mL/L to about 2.40 mL/L polysorbate 20;
about 0.04 ppm to about 50 ppm ProClin® 300;
about 9 mL/L to about 200 mL/L methanol; and
water,
wherein the rinse solution has a pH of from 6.6 to 7.0.
37 . A specimen preparation kit, comprising:
(a) a cytological fixative solution comprising
about 0.5 g/L to about 5.0 g/L Azure B;
about 0.5 mL/L to about 2.0 mL/L polysorbate 20;
about 5 mL/L to about 50 mL/L ethylene glycol;
about 0.10 g/L to about 10 g/L HEPES sodium salt; and
methanol;
(b) a cytological staining solution comprising
about 0.5 g/L to about 5.0 g/L Eosin Y;
about 5 mM to about 250 mM bis-tris buffer;
about 0.5 mL/L to about 2.0 mL/L polysorbate 20;
about one g/L to about 20 g/L sodium chloride;
about five mL/L to about 50 mL/L ethylene glycol;
about 0.2 ppm to about 50 ppm ProClin® 300;
acetic acid; and
water,
wherein the solution has a pH of from 5.8 to 6.2;
(c) a cytological staining solution of claim 19 ; and
(d) a rinse solution of claim 36 .
38 . A method of preparing a specimen, comprising:
treating the specimen with a cytological fixative solution comprising
about 0.5 g/L to about 5.0 g/L Azure B;
about 0.5 mL/L to about 2.0 mL/L polysorbate 20;
about 5 mL/L to about 50 mL/L ethylene glycol;
about 0.10 g/L to about 10 g/L HEPES sodium salt; and methanol;
treating the specimen with a cytological staining solution comprising
about 0.5 g/L to about 5.0 g/L Eosin Y;
about 5 mM to about 250 mM bis-tris buffer;
about 0.5 mL/L to about 2.0 mL/L polysorbate 20;
about one g/L to about 20 g/L sodium chloride;
about five mL/L to about 50 mL/L ethylene glycol;
about 0.2 ppm to about 50 ppm ProClin® 300;
acetic acid; and
water,
wherein the solution has a pH of from 5.8 to 6.2;
treating the specimen with a cytological staining solution of claim 19 ; and
treating the specimen with a rinse solution of claim 36 .