1. Field of the Invention
The present invention relates generally to immuno-histochemistry (IHC) staining of tissue samples and more specifically to a slide pocket that enables randomly accessing and individually processing a single tissue slide within an automated IHC system.
2. Discussion of Background Information
Immuno-histochemistry staining (IHC staining) requires several processing sequences such as, for example, (a) deparaffinization and tissue hydration; (b) target or antigen retrieval, (c) immuno-histochemical staining, (d) counter staining and (e) tissue dehydration. Several instruments have automated the process of IHC staining. In all cases, various instrument resources (e.g., reagents, heat, pipettes, physical locations for slides, wash buffers, etc.) are required for automation. The process of automation requires either (a) the sample (tissue on a slide) to be brought to those resources or (b) resources to be brought to the sample.
All current automated IHC systems require placing samples in the area where samples are processed, i.e. where processing steps can occur. Typically, samples are first loaded in batches of slides in a rack, tray, drawer or some physical fixture that enters an area where the system applies batch processing steps. Batch processing requires treating all slides in a single batch with the same reagents in accordance with a single set of batch processing steps. This type of batch processing results in a plurality of sample slides (e.g., all those included in a single rack or tray) being immersed or contacted with one reagent volume thereby creating the possibility of cross-contamination and false signaling. Current automated IHC staining systems therefore preclude random processing of individual slides in accordance with a specific, individualized sequences of processing steps, specific processing step durations customized for each IHC slide sample, and/or application of slide-specific reagent types, temperatures, and concentrations.
A need therefore exists for a slide pocket that enables individual tissue slide processing and treatment with multiple reagents of varying concentration and temperature within an automated IHC system.
One embodiment of the slide pocket includes a first shell portion having a slide well with a depth suitable for accepting the insertion of a sample-containing portion of a slide thereby positioning a sample affixed to the sample-containing portion of the slide within the slide well. In one embodiment, the slide well has a plurality of beveled interior walls, or wall faces, the beveled portions functioning to prevent the sample affixed to the sample-containing portion of the slide from contacting any surface of the slide well during insertion. In the present embodiment, the slide pocket has a second shell portion, mateable in fluid tight engagement with the first shell portion. The second shell portion has a slide well of depth suitable for accepting the insertion of a portion of the slide that is not a sample-containing portion of the slide.
In the present embodiment, the first and second shell portions, when in mated configuration, define a single continuous slide chamber defined by the slide wells of the first and second shell portions, a fluid input channel and fluid input port in communication with the single continuous slide chamber, and a fluid output channel and fluid output port in communication with the single continuous slide chamber.
In one embodiment, the plurality of beveled interior walls are sloped at a range of angles between 1 and 5 degrees relative to the front and back main faces of the slide. In another embodiment, the plurality of interior walls defines at least one notch.
In one embodiment, the first and second shell portions are monolithic. In another embodiment, the first and second shell portions are multi-component elements.
In one embodiment, the slide pocket has an intermediary fluid dispersal slot disposed between the input channel and the continuous slide chamber. In one embodiment, the intermediary fluid dispersal slot defines a pyramid (i.e. triangularly-shaped cavity) descending from the fluid input channel.
In one embodiment, the slide pocket is manufactured from a material capable of withstanding fluid temperatures of 0 degrees to 100 degrees C. In one embodiment, the slide pocket is injection molded. In one embodiment, the slide pocket is manufactured from a chemically inert plastic material. In one embodiment, the slide pocket is manufactured from one of Ultem®, urethane, or Vextra®. In one embodiment, the slide pocket of claim 1 wherein the slide pocket is grown from a ceramic material. In one embodiment, the slide pocket of claim 1 wherein the slide pocket is manufactured from stainless steel. In one embodiment, the slide pocket is of a hybrid construction of stainless steel and a non-metallic substance.
In one embodiment, the ratio of slide volume to volume of liquid in the slide chamber with the slide inserted is in a range of 1:0.3 to 1:10.
In one embodiment, the slide pocket has an integrally formed arm extending from an external surface for retaining and aligning the slide pocket within an automated slide processing system. In one embodiment, the slide pocket has one or more sealing gaskets disposed between the first shell portion and the second shell portion.
In one embodiment, the slide pocket has one or more slide standoffs positioned on the interior bottom surface of the continuous slide chamber for supporting an inserted slide above the interior bottom surface and enabling fluid flow around the bottom edge of the slide.
One will better understand these and other features, aspects, and advantages of the present invention following a review of the description, appended claims, and accompanying drawings in which:
The individual slide treatment pocket solves the problems left unaddressed by standard bulk processing IHC stainers and enables treating an individual slide with specific types and concentrations of reagents on demand and in specific volumes while eliminating the risk of cross-contamination of slide tissue samples. The slide pocket enables individual slide processing and treatment with reagents of varying concentration and temperature within an automated IHC system. The slide pocket is designed for use through the various processing steps of an IHC system such as, for example, dewaxing, alcohol dehydration, treatment with preheated antigen retrieval buffer, and rehydration with a series of alcohol (etOH), water and buffer solutions.
Turning now to specific slide pocket elements,
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In an alternate embodiment shown in
In all embodiments, such as that shown in
In all embodiments, the slide pocket 10 is designed for withstanding exposure to extreme temperature ranges and fluctuations and exposure to various chemistries. The slide pocket 10 is designed for washing and reuse such that cross contamination of samples is eliminated. The slide pocket 10, therefore, is manufactured from one or more inert materials capable of withstanding various chemistries and temperatures. In embodiments, the slide pocket 10 is manufactured from one or more inert materials, such as chemically inert plastic, ceramic, or stainless steel. In one embodiment, the slide pocket 10 is grown from a ceramic material. In another embodiment, the slide pocket is injection molded from a chemically inert plastic such as, but not limited to, Ultem®, urethane, or Vextra®, and the slide pocket 10 is capable of withstanding temperatures ranging from 0 degrees Celsius to 100 degrees Celsius without any degradation or catastrophic failure, even under rapid fluctuations in temperature. In another embodiment, the slide pocket 10 is manufactured from stainless steel panels having injection molded sidewalls. This hybrid material embodiment increases the rate of heat transfer during a thermal quench within the continuous slide chamber 300. In one embodiment, such as that shown in
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Additionally, the plurality of beveled sidewalls 150 extend far enough such that a range of sizes of slides 400 are accommodated by the slide well 105 without touching the tissue sample on the slide 400 and while enabling effective fluid saturation and processing of the smallest slide and the largest slide. The slide pocket 10 therefore accommodates slides ranging in at least width and thickness dimensions, for example, dimensions ranging from those of the Superfrost® Plus® slides to ColorFrost® Plus® slides. In an alternate embodiment, the plurality of beveled interior walls 115 may be replaced by or supplemented with a plurality of notches (not shown) that accommodate specific ranges of slides and result in a small peripheral surface area of the main face 410 of a slide 400 touching interior surfaces of the slide well 105.
The embodiment of the slide pocket 10 of
The angle α is determined in part by the horizontal and rotational distances across which the smallest and largest slides will lean in the pocket. This consideration is significant in the context of an automated handling system. For example, in one automated system (not shown), a robotic arm moving about the system contains a gripper that inserts and removes a slide 400 vertically from the slide pocket 10 for delivery to various system processing areas. The gripper contact area has a tolerance within which the slide 400 must mate during retrieval. If the angle α is too large and the slide 400 is flopped forward or backward too far, the gripper will not align properly with the edges of the slide 400 for secure engagement. The angle a is toleranced such that the smallest slide 400, when tilted forward or backward to greatest extent, will still align with the contact area of the gripper such that the gripper securely engages the outer edges of the slide 400. In preferred embodiments, the gripper has a relative moh value greater than that of a glass slide 400 for secure and safe handling.
Additionally, in one embodiment (not shown), the gripper attaches to a robot arm via a series of gearing that enables the gripper to raise and lower a slide 400 vertically into and out of a slide pocket 10 and also rotate the slide 400 to a horizontal position for insertion into a horizontally oriented IHC stainer tray. In this embodiment, the robotic arm is operated by a computing system designed to schedule processing of each individual slide and therefore the movement of the robotic arm. Additionally, the robotic arm only need move in an X-Y plane because the geared gripper is designed for movement along a Z-axis as well rotational movement in a Z-Y plane about an X axis, where the X axis is parallel to the length of the automated system, the Y axis is parallel to the width of the system, and the Z-axis is parallel to the depth of the system.
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It is noted that the foregoing examples have been provided merely for the purpose of explanation and are in no way to be construed as limiting of the present invention. While the present invention has been described with reference to an exemplary embodiment, it is understood that the words, which have been used herein, are words of description and illustration, rather than words of limitation. Changes may be made, within the purview of the appended claims, as presently stated and as amended, without departing from the scope and spirit of the present invention in its aspects. Although the present invention has been described herein with reference to particular means, materials and embodiments, the present invention is not intended to be limited to the particulars disclosed herein; rather, the present invention extends to all functionally equivalent structures, methods and uses, such as are within the scope of the appended claims.
The following three Examples relate to IHC staining carried out manually in Sakura baskets, and to IHC staining carried out using a slide pocket of the present invention. Example 1, for example, demonstrates surprising results in connection with a thermal quench step added following antigen retrieval pretreatment. Example 2 presents experiments suggesting that flash evaporation resulting from the brief exposure of a slide, following 98° C. treatment, to atmospheric conditions, may have resulted in peripheral tissue drying resulting in false negative results. Example 3 provides data supporting a conclusion that the use of the slide pocket tends to eliminate the type of false negative resulting from flash evaporation as reported in Example 2, when compared with otherwise identical experiments carried out in Sakura baskets. In addition to the benefits associated with a thermal quench step, the data taken as a whole demonstrate that the slide pocket is effective in eliminating temperature changes and drying caused by exposure of the sample to the environment.
This Example demonstrates that a cold flush step (i.e., thermal quenching) adds value to overall staining quality (after pretreatment) for antibody staining.
Design: 20 sequential tonsil sections from the same block (from a previously fixed and paraffin embedded sample) were pretreated with citrate buffer in a PT module, an automated slide dewaxing and processing device (Lab Vision Corporation, Fremont, Calif., a part of Thermo Fisher Scientific, Inc.). Five slides were processed per our current protocol (temperature ramped down to 65° C.; “° C.” refers to degrees centigrade) and served as the control; the remaining slides were processed with different post pretreatment methods in sets of 5. First set was shocked with cold buffer (3° C.) for 2 min; the second set was shocked with room temperature (RT) buffer (about 20° C.) for about 2 min; and the third set was shocked with 65° C. buffer. The samples were stained for the presence of CD68, a standard macrophage and monocyte marker. Staining was performed with anti-CD68 antibody at 1:6000 as described below. For each set of slides a control without antibody was included. The slides were processed in Sakura baskets. The procedure was as follows.
Fill PT Module with Buffer
Deparaffinize Slides and Hydrate to Water
Place Slides in Pre-Heated PT Module
Cool Slides in Buffer
Immunostain Slides
An exemplary staining protocol is as follows. Many satisfactory staining protocols are known to those of ordinary skill in the art and/or are provided by the supplier of the antibody being used. An automated system, as indicated above, or manual processing may be used.
Primary Antibody Use
General, Exemplary Detection Kit Staining
Conclusions:
Summary: The results indicate that the cold flush is useful to improve staining quality in instances where an antibody gives unacceptable background staining.
This Example provides further data with regard to utilization of a cold flush with samples after pre-heating with regard to slides processed in Sakura baskets. The experiments are summarized below.
Purpose: To determine cold flush temperature limits
Design: Six sections each from 3 different tissues and a UTA microarray were dewaxed and rehydrated per the protocol given in Example 1. The slides were all pretreated in 98° C. buffer for 20 min followed by placing in 3° C. or 10° C. or 15° C. baths for 2 min each. Unlike the previous example, the slides were manually processed up to this point in the procedure. The slides were then stained using the Quanto detection system on the Autostainer™ as detailed in Example 1.
CD117 was identified as a candidate for this experiment because the antibody used results in high NSS. The goal of the experiment was, in part, to determine if the cold flush reduces this background. In the results below, the antibody bound to the colon core and breast cancer in a non-specific manner. Similar results were seen in the samples tested in the GIST array. MART-1 was selected for use on the UTA array because melanoma cores were present on the UTA. This combination of samples and antibodies achieved several levels of staining intensities.
Observations and Conclusions:
Next Steps:
Two 2 sets of experiments were performed to determine the cut off temperature for the cold flush. One set of data were obtained using the “slide pocket” as the slide carrier, while the second set of data were obtained by using the Sakura basket as the slide carrier. The slide pocket is a self contained device designed for optimal slide processing with minimal reagent use and allowing for rapid reagent flushing and rapid sample temperature changes.
Purpose: Determine cold flush cutoff temperature
Design: #1: Ten sequential tonsil sections were placed in a slide pocket (3 batches) in the PT module when the temp reached 98° C. After 20 min in the 98° C. buffer, 2 slides each were shocked in 3° C., 10° C. and 15° C. baths. The 3° C. shock was performed for 2 min. The 10° C. and 15° C. shocks were performed for either 2 min or 5 min. Two control slides were processed in the slide pocket in the PT module for 20 min and ramped down to 65° C.
#2: Eighteen sequential tonsil sections were processed in the Sakura baskets in the PT module after the buffer reached 98° C. and were held for 20 min. Then 6 slides each were shocked in 3° C. or 10° C.; 3 each for 2 min and 5 min respectively. Six other slides were shocked at room temperature (about 17° C.) for an hour. Three slides were processed as controls per the standard procedure as described in Example 1. The control slides were ramped down to 65° C. in the PT module.
The slides from designs #1 and #2 were stained on the Autostainer with CD68 antibody at 1:6000 using the Quanto detection system (see, Example 1).
3+/−
3+/−
“X” indicates slides were not treated under the conditions listed on the table. Focal neg. indicates that the samples displayed false negative rings, as described above in Example 2. Staining was graded on a scale of 1-4 with 1 indicating little or no staining and 4 indicating the strongest staining. The designation “3+/−” is indicative of staining that is slightly less intense than the designation “3+”. The designation RT (room temperature) indicates a temperature of about 20° C.
Conclusions:
Thus, when taken as a whole the results of these three Examples demonstrate that the slide pocket is effective in eliminating temperature changes and drying caused by exposure of the sample to the environment and the slide pockets are effective in providing for higher quality staining. For example, Example 1 shows that the use of thermal quenching is effective in decreasing background staining. Examples 2 and 3 demonstrate that the use of the slide pocket eliminates the false negative ring artifact that is observed when slides are processed in a manner that permits atmospheric exposure of the tissue sample.
Number | Date | Country | |
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61592880 | Jan 2012 | US |