Classifications

• • 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
• • 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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6827—Total protein determination, e.g. albumin in urine
  • G01N33/6839—Total protein determination, e.g. albumin in urine involving dyes, e.g. Coomassie blue, bromcresol green
• • 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
  • G01N2001/302—Stain compositions

Definitions

• H and E staining technique is the most commonly used histological technique for the visualization of pathology in tissue specimens.
• a typical H and E staining system is composed of solutions comprising aluminum based hematoxylin, eosin, a differentiating solution, and a bluing agent.
• Hematoxylin is a natural dye that when complexed with aluminum ions produces a positively charged molecule that binds to deoxyribonucleic acid to produce a purple coloration of cell nuclei.
• Differentiation solutions typically are lightly acidic solutions that sharpen the contrast of the stained slide by removing excessive background staining from the tissue and slide.
• Bluing agents are solutions of a basic pH that when applied to stained specimens modify the structure of the bound hematoxylin to produce a vibrant blue/purple coloration.
• Eosin is a negatively charged dye that binds to positively charged proteins within the cytoplasm and connective tissue to produce a pink/orange coloration that contrasts with the blue/purple coloration of the nuclei.
• H and E staining is most commonly performed in a "batch" format wherein automated stainers typically contain a series of staining reagent containers and utilize a robotic arm for the sequential movement of slides/racks from one container to another.
• a rack or holder is capable of carrying 20 to 30 slides although some staining instruments incorporate racks that hold fewer or greater numbers of slides per rack and in the case of at least one stainer, multiple racks may be places into a staining vessel simultaneously.
• Software controlling an automated stainer allows the operator to program the duration of each of the steps as well as the sequence of the steps. While the batch format of staining is simple, economical, and relatively rapid, there are drawbacks with the technique.
• carry-over negatively impacts the performance, functionality, stability, predictability, and capacity of the staining system. Although a single instance of carry-over may result in such negative impacts, the negative impacts of carry-over are especially problematic over the course of multiple staining runs as the negative impacts build or are amplified as the result of the cumulative effects of repeated carry-over.
• Certain embodiments of the present invention provide for solutions of a defined composition useful in a staining protocol when used at certain points of the staining protocol.
• the formulations of these solutions are such that carry-over of the solutions will not negatively impact, or preferably, will stabilize or favorably modify staining reagent solutions coming in contact with solutions defined herein.
• solutions are buffered to maintain a specific pH that when carried-over-such as carried-over into hematoxylin-will not significantly influence the pH of the next staining reagent.
• Certain embodiments of the invention are drawn to methods of reducing the undesirable impact of solution carry-over in an H and E staining system.
• defined solutions are used at various steps of the staining protocol, such as before certain staining reagent steps, to reduce the undesirable impact of carry-over.
• certain embodiments of the invention are drawn to a pH buffered solution comprising an organic acid and a polyhydroxy alcohol.
• the pH buffered solution has a pH buffering range that comprises from about pH 2.0 to about pH 5.0.
• the pH buffered solution has a pH buffering range of from about pH 1.0 to about pH 7.0, or from about pH 1.0 to about pH 6.0, or from about pH 1.0 to about pH 5.O., or from about pH 2.0 to about pH 7.0, or from about pH 2.0 to about pH 6.0.
• such pH buffered solution has a pH buffering range of from about pH 2.0 to about pH 5.0.
• the pH buffered solution may comprise from about 5% to about 30% (v/v) of a polyhydroxy alcohol selected from the group consisting of ethylene glycol, propylene glycol, glycerin, and polyethylene glycol and may comprise from 10 mM to about 100 raM of an organic acid selected from the group consisting of malic acid, citric acid, maleic acid, acetic acid, and tartaric acid.
• the pH buffered solution may also comprise an antimicrobial agent that is effective over at least a pH range of from about pH 2.0 to about pH 5.0.
• Certain embodiments of the invention are drawn to a solution with a pH of from about pH 2.0 to about pH 5.0 comprising aluminum salts and from about 5% to about 30% (v/v) of a polyhydroxy alcohol selected from the group consisting of ethylene glycol, propylene glycol, glycerin, and polyethylene glycol.
• Certain embodiments of the invention are also drawn to a method of staining a tissue specimen with hematoxylin, the method comprising the steps of (i) contacting the specimen to a defined solution of the invention and (ii) transferring the specimen to a hematoxylin staining solution. Additionally, the method may comprise the steps of rinsing the specimen with water and then contacting the specimen with the buffered solution.
• kits for performing hematoxylin and eosin staining comprising a defined solution of the invention and at least one hematoxylin and eosin staining reagent selected from the group consisting of a hematoxylin solution, an eosin solution, a differentiating solution, and a bluing agent solution.
• Figure 1 shows the steps of a typical hematoxylin and eosin staining protocol.
• Figure 2 shows changes in the pH of a hematoxylin solution during the sequential staining of 2,500 slides with specimens. Staining of multiple slides results in an increase in the pH of the hematoxylin solution (solid line). Placement of a buffered solution of defined composition prior to the hematoxylin prevents an increase in pH of the
• Figure 3 shows changes in the quality of hematoxylin staining of specimens following the sequential staining of 2,500 slides. Increased cytoplasmic background staining and loss of detail is present in the 2,500 th slide as compared to the first slide.
• Figure 4 shows consistent hematoxylin staining observed in the first and 2,500 th sequentially stained specimens. Slides were stained with a protocol using a buffered solution of defined composition of the invention between the tap water rinse and the hematoxylin solution.
• a "staining reagent” refers to any reagent used in a histological staining protocol. Where it is necessary to understand the invention by specifically referring to "a reagent that is a stain” versus "a reagent in the staining process that is not a stain,” such a distinction is explicitly made.
• a "working strength" or “ready-to-use” solution is a solution wherein the components in the solution are at the desired concentration for use in the methods of the present invention. "Ready-to-use” may also imply that the solution does not require any additional manipulation on the part of the end user such as filtration, skimming, and/or mixing.
• xylene substitutes which are in widespread use include two classes: terpenes and aliphatic solvents.
• terpenes include limonene and turpentine.
• tap water is most economical to use as a rinse step in high capacity H and E staining protocols.
• water refers to tap water, but also covers distilled or deionized water or aqueous solutions used to treat or rinse specimens in an H and E staining system.
• H and E staining is a widely used staining method for histological purposes.
• One of skill in the art will recognize that many variations and protocols for H and E staining have been developed and where the present invention is applicable, the present invention is not limited to any specific variation, protocol, system, or the like.
• a typical H and E staining system may be defined as composed of four core staining reagents: hematoxylin,
• a typical, representative H and E staining protocol comprises the steps of (i) deparaffinization (using xylenes or xylene substitutes), (ii) rehydration (using alcohols), (iii) water rinse, (iv) hematoxylin, (v) water rinse, (vi) differentiator, (vii) water rinse, (viii) bluing, (ix) water rinse, (x) alcohol, (xi) eosin, (xii) dehydration (using alcohols), and (xiii) clearing (using xylenes or xylene substitutes) (Sheenan, D.C. and Hrapchak, B.B.: Theory and Practice of Histotechnology 2 nd ed.
• a step of using a defined solution immediately precedes a step in the H and E staining protocol.
• the composition of the solution is dictated by the staining reagent that the solution is designed to protect from carry-over.
• a step of using a defined solution of the invention immediately precedes a core staining reagent step.
• a step of using a defined solution immediately precedes the hematoxylin step. In certain embodiments of the invention, a step of using a defined solution immediately precedes the differentiator step. In certain embodiments of the invention, a step of using a defined solution immediately precedes the bluing reagent step. In certain embodiments of the invention, a step of using a defined solution immediately precedes the eosin step.
• a solution with a defined composition is placed between a water rinse step and the hematoxylin solution.
• the rinse water is tap water.
• the defined solution mitigates several adverse effects of the water on the hematoxylin solution.
• the volume of components for example, ions, chlorine, etc.
• the addition of a buffer to the solution reduces the capacity of the water to increase the pH of the hematoxylin ( Figure 2). This is advantageous because an increase in the pH of the hematoxylin results in background staining of tissues and loss of staining detail ( Figures 3 and 4).
• the defined solution may be formulated to enhance the stability of a hematoxylin exposed to carry-over of the solution.
• the addition of a polyhydroxy alcohol to the solution to be carried over to the hematoxylin can reduce the likelihood of precipitation of the hematoxylin and can provide value to the user as filtration of the hematoxylin will not be necessary.
• a step of "using" a solution means exposing a sample-for example a tissue specimen mounted on a slide-to a solution.
• a sample may be exposed to a solution, for example, by dunking or immersing it in the solution.
• a sample may be exposed to a solution, for example, by passing the solution over the sample, such as by passing a solution over a slide holding a sample.
• a sample may be exposed to a solution, for example, by dropping or spraying the solution onto a sample.
• One of skill in the art will recognize that there may be various alternative methods of exposing a sample to a solution during the staining process, and that the method and solutions of the present invention are not limited to any particular method of exposing a sample to solution.
• One aspect of the present invention provides for solutions of defined composition for mitigating the effects of water or staining reagent carry-over in a histological staining protocol.
• the defined solution is a buffered solution comprising an acid, the buffered solution being capable of buffering from between about pH 2.0 to about pH 5.0.
• the acid has a pKa of from about 2.0 to about 5.0.
• the concentration of the acid in the solution is from about 10 mM to about 100 mM.
• the concentration of the acid in the solution is from about 20 mM to about 100 mM.
• the concentration of the acid in the solution is from about 10 mM to about 50 mM or from about 20 mM to about 50 mM.
• the acid is an organic acid.
• the buffered solution comprises a polyhydroxy alcohol at a concentration of from about 5% to about 30% (v/v).
• polyhydroxy alcohols include, but are not limited to, ethylene glycol, polyethylene glycol, propylene glycol, and glycerin.
• the solution comprises both an acid and a polyhydroxy alcohol in the amounts and as defined herein.
• the buffered solution is a solution comprising 20 mM tartaric acid and 15% (v/v) ethylene glycol and the solution pH is adjusted to a pH of 3.3.
• pH is adjusted with sodium hydroxide (NaOH).
• the defined solution is an unbuffered solution with a pH of from about pH 2.0 to about pH 5.0 comprising aluminum salts.
• aluminum salts include, but are not limited to, aluminum ammonium sulfate [A1NH 4 (S0 4 ) 2 ], aluminum sulfate [A1 2 (S0 4 ) 3 ], and aluminum potassium sulfate A1K(S0 4 ) 2 .
• the unbuffered aluminum salt solution also comprises a polyhydroxy alcohol as described herein at a concentration of from about 15% to about 30% (v/v).
• the defined solution is a slightly modified or non-modified formulation of the staining reagent used as the differentiator.
• differentiator is a solution buffered with any acid, where the acid has a pKa of from about 2.5 to about 4.0 and is capable of buffering between a pH of from about pH 1.5 to about pH 5.0.
• the acid is an organic acid.
• useful organic acids include, but are not limited to, malic acid, citric acid, fumaric acid, maleic acid, acetic acid, and tartaric acid.
• an antimicrobial agent may be added to inhibit microbial growth.
• the antimicrobial agent is one that is effective over at least a pH range of from about pH 2.0 to about pH 5.0.
• Representative examples of microbial agents include sodium azide, PROCLI 300 ® , PROCLIN 150 ® , PROCLIN 200 ® , and PROCLIN 950 ® .
• the solution contains from about 0.03% to about 0.04% (v/v) PROCLIN 300 ® (Sigma Aldrich, St. Louis, MO) to inhibit microbial growth. It is to be understood that example product or trade names known to those of ordinary skill in the art are provided for illustrative purposes only and are not meant to be limiting of the broader genus of reagents described.
• the defined solutions such as the buffered solutions, of the invention will be provided as a working strength or ready-to-use solution for H and E staining protocols.
• concentrated solutions of two or more components may be prepared wherein a user may add water and/or other components (such as an antimicrobial) to the defined solution to prepare a working strength or ready-to-use solution with the formulations described herein.
• the present invention is also drawn to concentrated solutions that can be used to prepare the defined solutions of the invention. Given the amounts of components in the defined solutions provided, it is within the skill of one of ordinary skill in the art to determine what the amounts of components in a
• concentrated solution must be to provide for a working strength or ready-to-use solution. For example, if the concentrate is to be diluted 1 : 1 with water, then the concentration of an acid, polyhydroxy alcohol, etc., would be twice what the desired final concentration would be. V. Methods of Mitigating the Effects of Carry-Over
• One aspect of the present invention provides for methods of mitigating the effects of water or staining reagent carry-over in a histological staining protocol.
• such methods utilize a defined solution as a step in the staining process between two staining reagent steps or between a water rinse step and a staining reagent step.
• the samples may also be immersed in the defined solution.
• staining protocols where samples are contacted with staining reagents in a manner other than immersion, the samples may be contacted with the defined solution in a likewise manner. It is understood, however, that the manner in which the samples are contacted with the staining reagents does not limit the manner in which the sample may be contacted with the defined solution within the same staining protocol.
• hematoxylin and differentiator solutions the detrimental effects of carry-over are greatest on the hematoxylin and differentiator solutions, but may affect all stains/reagents in a staining system.
• Hematoxylin and the differentiator follow water rinses in a typical H and E staining schedule.
• the performance of these two solutions is highly dependent upon pH and the introduction of water to these solutions results in an increase in pH.
• One effect of increased pH on these solutions is an increase in the background staining of hematoxylin.
• a sample is contacted with a defined solution immediately before being contacted with a hematoxylin staining solution.
• the sample is contacted with a defined solution immediately after being rinsed in water and immediately before being contacted with a hematoxylin staining solution.
• the pH of the hematoxylin solution will increase less than 0.30 pH units when exposed to a buffered solution carried over from prior immersion of the specimen in the buffered solution.
• the pH of the hematoxylin solution will increase less than 0.20 pH units when exposed to a buffered solution carried over from prior immersion of the specimen in the buffered solution.
• a sample is contacted with a defined solution immediately before being contacted with a differentiator solution.
• the sample is contacted with a defined solution immediately after being rinsed with water and immediately before being contacted with a differentiator solution.
• Use of the described methods may allow for up to 2,000 standard specimen slides to be processed under standard H and E staining procedures before new reagents are substituted. In certain embodiments, use of the methods may allow for up to 2,700 slides to be processed before new reagents are substituted. Further, in certain embodiments, use of the methods may allow for up to 3,000 slides to be processed before new reagents are substituted. It is understood by those of skill in the art that the capacity to process a certain number of specimen slides refers to the capacity to process such a number without observing an adverse effect on staining that would be unacceptable for the purpose for which the slides are being stained. It is further contemplated that use of the reagents and methods of the invention used in combination with mechanical methods that reduce, but do not completely eliminate carryover, will also significantly enhance the number of slides that may be stained using a given set of reagents.
• Methods of the invention may also include the step of diluting a concentrated solution, such as diluting with water, to a final working strength or ready-to-use defined solution.
• Methods of the invention may also include the step of adding a component, such as an antimicrobial, to prepare a ready-to-use defined solution. This step would be performed before using the defined solution in an H and E staining protocol.
• kits for performing H and E staining comprise at least a defined solution that mitigates the effects of solution carry-over.
• Useful defined solutions are described in detail in this application and include buffered and unbuffered solutions.
• Representative kits of the invention may also comprise one or more staining reagents or other components used for H and E staining. Examples of staining reagents used for H and E staining include hematoxylin solution, eosin solution, a
• the kit comprises at least the five reagents: (1) hematoxylin solution, (2) eosin solution, (3) a differentiating solution, (4) a bluing agent solution, and (5) a defined solution that mitigates the effects of solution carry-over.
• kits for performing H and E staining include using a defined solution that mitigates the effects of carry-over as described in detail in this application.
• the use of a kit provides numerous benefits. Current H and E staining procedures and reagents are subject to the negative effects of solution carry-over, often necessitating that the solutions be changed at different rates.
• the inclusion of a defined solution that mitigates the effects of solution carry-over in a kit provides for a predictable lifespan of all the kit reagents that is consistent, such that the kit reagents require changing at the same time.
• kit reagents may allow for up to 2,000 standard specimen slides to be processed using standard H and E staining procedures before new reagents are substituted. Or use of the kit may allow for up to 2,700 slides to be processed before new reagents are substituted. Or use of the kit may allow for up to 3,000 slides to be processed before new reagents are substituted.
• composition places between the tap-water rinse and the hematoxylin step (Table 2). This solution was designed to mitigate the effects of carry-over of the tap-water into the hematoxylin. This solution like the hematoxylin, differentiator, bluing agent, and eosin was not changed during the depletion study. As with the study described above, staining of control tissue specimens on multi-tissue control slides (functional staining) and determination of the pH of the hematoxylin were performed at 150 slide intervals.
• Figure 2 demonstrates the increase in pH of the hematoxylin that occurs as slide racks with slides carry over tap-water to the hematoxylin.
• the placement of a buffered reagent of defined composition between the tap water and the hematoxylin displaces the tap- water from the slides prior to movement into the hematoxylin solution.
• the pH of the hematoxylin remains stable.
• Table 1 shows a standard H and E protocol performed on a LEICA ST-5010XL Automated Stainer.
• Table 2 shows an H and E protocol using a buffered solution of defined composition places between the tap water rinse step and hematoxylin step (Step 10 in Table 2) on a LE ® ST-5010XLTM Automated Stainer.
• Table 3 shows a representative example of a buffered solution of defined composition of the invention, such as for use immediately prior to contacting a specimen slide with hematoxylin solution.
• the pH is adjusted to 3.33 with 2N NaOH.

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