Frequently Asked Questions
How do we control the target density in the secondary arrays?
We use pure Rabbit and Mouse IgG proteins diluted with a non-reactive spacer protein to form the gradient density array(s). The individual protein primary dilutions are evaluated by their absorbance at 280nm. The data is then used to formulate the deposited protein array target mixtures, ensuring consistent performance between different IgG lots.
While the secondary gradient array mixtures can produce an absolute concentration scale it cannot account for the slide coating’s physical structure. All IHC slides that exhibit hydrophilic behavior and tissue retention through the antigen recovery processing have a porosity factor to some degree. For tissue sections this is of no consequence other than the slide coating being conformably compliant to the physical irregularities caused by the sectioning blade on the section’s surface and reagents spreading across the slide’s surface. However, for proteins, the porosity and wettability variables of the slide coating affects how much deposit volume will be required to fill the voids and still leave a sufficient layer on the surface. Thus, having tight control over the slide coating process is critical to ensuring uniformity of stain reactivity slide to slide. During manufacture of the PRS, we monitor the deposition of the targets for uniformity, culling out any that do not meet our criterion. The result is a tight distribution in the target reactivity slide to slide. The factor we cannot control is the precipitation capacity of the chromogen. However, for a single slide the ratio between primary and secondary stained target arrays will be tight to providing a relevant scale to apply against the tissue section.
How can a two density primary target pair be used with a secondary gradient array to form a primary gradient density measurement scale?
The primary & secondary array deposits all experience the same impact of the slide coating’s porosity and wettability when being made. So, while there will be differences slide to slide the relationship between the primary and secondary targets remains the same because the target materials are all adjusted to a fixed standard. The primary targets represent the cumulative experience from all the stain reagents while the secondary targets represent only the cumulative secondary reagents experience.
Calibration of the secondary target array materials is easily done by UV absorbance measurement during formulation of the target mixtures. Such calibration enables the gradient density array to have a known stain intensity profile independent of the secondary stain method used. The only exception occurs when the chromogen precipitation deposit covers the enzyme site stopping continued buildup of chromogen. The result is a stain intensity (light to dark) series incrementing with a known square law intensity sequence profile across the secondary array.
All target array series progress from low to high reactive density following a square-law profile.The primary target array has three members containing different, but known, densities of reactive antigens. Since the primary targets also experience the secondary stain processing and the primary target densities are known, then with the three data points an expanded antigen density scale can be mathematically constructed from the secondary array.
The primary purpose of the PRS is to objectively measure the IHC staining performance against known density targets. However, the PRS target data can provide a measurement baseline to apply to the co-resident tissue section through the use of digital imaging and post capture processing. For example:
- The digital image can adjust for maximal white to black expansion of the image because actual white and black target cells are incorporated within the target array. Typically, a stained result is mostly within the top 30% of stain density, effectively compressing the data. Having the black and white target cells as part of the slide enables correction in both the camera digitization and provides feedback to the light source to change intensity if the high end (dark) is within compression.
- And/or the image color density can be shifted to normalize the presentation to a consistent color palette.
- The image processing program can set to present the same image to different viewers adjusted for their optimal diagnostic decision making because the staining experience is captured in the PRS targets.
- Tele-diagnostic interpretation is now possible as every slide contains the information necessary to reconstruct the image presentation correctly on multiple screens with adjustment for individual users as well. Such capability allows the image to be shifted in color for what the observer finds best for them. In practice, they do this by changing the microscope lamp intensity and color. With digital imaging, such action can be applied at any time in the future without handicapping any other user.
- Regulatory approvals for digital imaging diagnostic pre-screening have been thwarted simply because the regulatory bodies have insisted that the scanner be knowledgeable of every processing step that each slide went through. That is simply not possible when the slide and any tracking data is independent of what the slide actually experienced. In a large respect, this mandate seeks to cover up the non-regulatory approved: processing, reagents, and staining technologies that exist today. In other words, the imaging system is the last one to hold the hot potatoes and thus becomes responsible for all that took place before it. The PRS, however, changes all this as it forever records the experience it went through to become a stained slide. Thus, the slide does not depend on a LIS other than to record what primary stain(s) were applied. At last the digital imaging pre-screening diagnostic are viable.
Can dual staining take place?
Of course!, Mating one primary stain to Mouse and the other to Rabbit and processing them sequentially with single species secondary stain kits having different substrate/chromogens allows for different colors to be produced that reflect one primary antibody vs. the other.
How do different secondary stain kits or lot codes even with a single kit source (primary and secondary) yield different results?
Most secondary stain kits support binding to both Rabbit and Mouse proteins. The exception to this is when dual detection staining is to be performed wherein one antigen is detected by a Mouse secondary stain and the other is detected by a Rabbit secondary stain. The reactivity of the secondary stain kit to the Rabbit or Mouse proteins is usually not supplied by the stain kit manufacturer. If both the anti-Mouse and anti-Rabbit proteins are monoclonal, the end stain density will be pretty even between the two animal sources. However, some kits use polyclonal anti-Rabbit and monoclonal Mouse. Because the polyclonal is 3-4 times bigger than the monoclonal version, the stain intensity will drop by the same factor as the enzyme site density has dropped by a similar ratio. The result is a significant stain density difference between monoclonal and polyclonal reagents.
The primary stain reagent is an antibody on Mouse, Rabbit, or Goat serum. Can its KD be used to determine the antigen density in the tissue section?
The primary stain reagent is obtained by inoculation of the host species with the antagonist antigen. The host responds by making antibodies on its blood serum. The density of antibody sites on the blood serum protein depends upon the animal’s genetic variation. Mouse has been bred to have a very narrow genetic variation while Rabbit is wider. The degree of consistent genetic replication between the donor animals directly impacts the antibody density on the blood serum. The variation is measured as KD, (the quantitative measurement of antibody affinity), and many primary stain reagent vendors provide the KD value for each lot code of their reagents. Knowing the applied density of antigen sites on the PRS primary targets, the KD of the primary stain reagent, and the PRS secondary target densities the nominal density of antigen sites on the co-resident tissue section can be determined from the calculated primary scale. The scale has its limitations which include:
- The recovery/damage effects of antigen recovering processing on the tissue vs. PRS targets. This will always be an unknown as the condition of the tissue section fixation is unknown. Obviously, there are two factors at play: fixation condition of the tissue section and the degree to which the antigen recovery processing method/buffer unmasks or damages the antigen sites
- If the wrong HIER buffer is selected relative to the tissue type and antigen being sought, the tissue antigen sites may not become exposed.
Can all secondary stain kits be supported by the PRS secondary gradient density target arrays?
The simple answer is yes. All the secondary stain kits bind to Mouse and/or Rabbit IgG proteins which are used in the PRS secondary gradient array.
Secondary stain kits providing amplification of the enzyme density to the target of interest have evolved into four different topologies:
- Avidin-Biotin complex (ABC)
- Labeled strepavidin biotin (LSAB)
- HRP polymer
The above all depend upon a primary antibody on an anti-Rabbit/Mouse protein (most often Goat anti-Mouse or Goat anti-Rabbit). The secondary stain starts with anti-Mouse or anti-Rabbit conjugated to the amplification pathway.
LSAB has been shown to increase detection sensitivity by 4-8x over ABC [see Giorno R (1984), Diagn Immuno 1.2:161-6]
In the case of the HRP polymer and EXPOSE the secondary stain is biotin free, thus avoiding background artifacts caused by endogenous biotin on the tissue section that would react with the avidin-biotin complex. Tissues with endogenous biotin include: kidney, liver, brain, intestines, colon, prostrate, and testes. EXPOSE eliminates the polymer backbone and attaches the HRP end groups to the detection antibody with long spacer arms, thus avoiding the polymer binding to itself.
- Tyramide signal enhancing (TSE)
This method binds additional biotin end groups, using a tyramide covalent bond, on and near a previously reacted target using a streptavidin – HRP complex. The additional biotin end groups are then exposed to the streptavidin – HRP complex to increase the enzyme action in precipitating the chromogen and thus the visual detection of the target presence. The primary Mouse/Rabbit antibody remains the same as used in the ABC, LSAB, and HRP Polymer staining processes.
Explain why Rabbit & Mouse can have such different reactivity behaviors
Historically, Mouse was the first to produce monoclonal antibodies while Rabbit remained polyclonal. At present Rabbit is usually monoclonal, but not with the breadth antibodies that Mouse supports. Polyclonal, being physically larger than monoclonal, is unable to attach to the target in as great a number as the monoclonal antibodies. All else being equal, the detection density is about 3-4x greater with monoclonal vs. polyclonal antibodies.
Rabbit serum has a greater variation in the density of antibody sites on the protein because the animals themselves have a larger genetic pool than Mouse. Thus, there will be a larger variation in monoclonal antibody site density on Rabbit vs. Mouse proteins.
Considerable effort has been applied to bring Rabbit to all monoclonal antibody status. Thus, for many primary antigen detection antibodies either Mouse or Rabbit is available in monoclonal status.
Why does the HIER temperature have so much impact?
The heat induced epitope retrieval, HIER, also termed heat released antigen recovery, HRAR, or simply antigen retrieval incorporates a heated caustic/acid buffer to decouple the formaldehyde cross-linking formed during the fixing of the biopsy sample tissue block. The target temperature is 95°C max, but better recovery occurs at 92-92°C and a longer exposure time. Most HIER systems are non-uniform in maintaining the HIER buffer temperature evenly across the active area of the slide. Thus, in some areas the formaldehyde is not fully removed keeping those antigen sites covered while in other areas the formaldehyde is fully removed but the buffer temperature and reactivity has had sufficient time to damage the antigen sites. Identifying the under or over exposed areas is not possible with the present staining technologies. The result is that diagnostic interpretation is potentially corrupted before diagnostic determination can even take place. PRS cannot report such spatial conditions because the targets are co-located in a small area of the slide. However, special target slides can be fabricated to support full spatial evaluation of the slide during antigen recovery.
The cumulative result - the IHC processing protocol is fixed so how is the PRS of value
The PRS contains both secondary and primary reactive targets.
The secondary targets react only with the secondary stain reagent series and are composed of a gradient density array of Mouse + Filler or Rabbit + Filler IgG proteins. The resultant secondary stain result is then the cumulative result of each of the secondary stain reagents and wash buffers.
The primary targets are composed of antigen clone peptide strands that are bound to a carrier. The peptide/carrier combo is then mixed with non-reactive carriers and the composite deposited onto the PRS. The primary target stained result is then the cumulative result of both the primary and secondary stain reagents and wash buffers.
The processing problems that can impact the staining performance include the following:
- Paraffin removal
- Antigen recovery process to expose the antigen sites
- Primary stain reagent
- Secondary stain reagent
For discussion, we will work backwards from the fully performing condition to defective step or reagent. If the secondary stain reagents are defective then there will be no staining anywhere on the slide. This failed condition can be caused by:
- PRS shield coating was not removed
- Destructive exposure during antigen recovery by excessive exposure at >95°C
- One of the secondary stain reagents failing completely
- Slide allowed to dry sometime following the shield coating removal
How stable is the PRS sealed and open box?
The PRS targets have a shelf life of one year in a sealed box condition and six months in open box condition. This is realized by the sealant coating applied over the targets to protect them from oxidation and microbial attack.
However, the adhesive coating on the slide has a much shorter open box shelf life. Due to the controlled hydrophilic condition the adhesive coating will attract and react with airborne contaminates, both organic and inorganic. Once, reacted the slide will lose its hydrophilic behavior and the adhesive capture strength will lose some strength due to loss of active capture sites.
The slides are packaged in a 25-slide plastic container. The container provides assurance that the slides will not rub against each other during shipping and damage the coating and PRS targets. The slides cannot stick together in the open box condition due to the inherent tendency to attract moisture from the air, and the container is free from outgassing. Many microscope slides are packaged in paper boxes. Unless the paper and label ink is acid free the paper box will release acid fumes that attack the slide coating and ruin the capture performance.Others use plastic boxes that can potentially outgas hydrocarbons (oils) from the plastic itself or from residual mold release.PRS is packaged in a manner to ensure no degradation effects can or will occur.