- Email: [email protected]
Automation of fluorescence in situ hybridization pretreatment: A comparative study of different sample types
Molecular Diagnosis Vol. 5 No. 3 2000
Automation of Fluorescence In Situ Hybridization Pretreatment: A Comparative Study of Different Sample Types KRISTINE JACOBSON, MS, ANTHONY THOMPSON, MA, GERRY BROWNE, BS, CHRISTINA SHASSERRE, MS, STEVEN A. SEELIG, MD, PhD, WALTER KING, PhD Downers Grove, Illinois
Background: In fluorescence in situ hybridization (FISH) applications, the efficiency of probe hybridization is greatly enhanced by treating the cell or tissue preparation with a variety of reagents that make the target permeable while preserving important morphological features. Pretreatment protocols can be very labor intensive, adding cost to the test. The automation of specimen pretreatment eliminates human variation in the procedure through standardization of such variables as treatment times and temperatures. Methods and Results: We developed an instrument, the VP 2000 Processor, that can process up to 50 specimens simultaneously under computer control. In this comparative FISH study of matched specimens, one set was processed according to the manual pretreatment protocol and compared with specimens processed by the VP 2000. Processed specimen types included paraffin-embedded breast tissue, uncultured amniocytes, bone marrow, peripheral-blood lymphocytes, and uroepithelial cells recovered from urine. Data show equivalent or brighter and more specific overall signal quality compared with matched manually executed controls. Conclusions: The automation of sample pretreatment for FISH provides a superior, more consistent level of performance than the manual format. Key words: fluorescence in situ hybridization, centromeric enumeration probes, locus-specific identifier probes, chromosome painting probes.
before FISH. The amount of labor required varies with the type of specimen, but it can be extensive with certain specimen types, such as paraffinembedded tissue [1,2]. We have developed an instrument, the VP 2000 Processor (Vysis, Inc, Downers Grove, IL), that automates a variety of pretreatment protocols on 50 specimen slides at the same time. Together with the HYBrite (Vysis, Downers Grove, IL), which automates denaturation and hybridization, hands-on processing time for directly labeled FISH probe assays is reduced 57% to 90%. Pretreatment protocols for paraffin-embedded tumor sections, uncultured amniocytes, bone marrow, uroepithelial
Fluorescent in situ hybridization (FISH) technology is becoming more widely used in today’s pathology laboratory. Despite the performance advantages in specificity and sensitivity, FISH has remained in a manual-assay format. A significant fraction of the labor required for optimal FISH performance involves the pretreatment of specimens From Vysis, Inc, Downers Grove, IL. Reprint requests: Walter King, PhD, Director, Assay Development, Vysis, Inc, 3100 Woodcreek Dr, Downers Grove, IL 60515. Email: [email protected] Copyright © 2000 by Churchill Livingstone威 1084-8592/00/0503-0007$10.00/0 doi:10.1053/modi.2000.9731
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210 Molecular Diagnosis Vol. 5 No. 3 September 2000
cells, and cultured lymphocytes are preprogrammed into the instrument software. The instrument has been validated in pretreatment protocols for several specific FISH assay specimen types: Vysis PathVysion (Vysis, Inc, Downers Grove, IL) (c-erb B2 [HER-2] and chromosome 17 enumeration probe [CEP 17] on paraffin-embedded breast tumor sections), the AneuVysion Assay (test for common prenatal aneuplodies on uncultured amniocytes), CEP 8, CEP 12, and LSI bcr/abl for leukemia diagnosis on bone marrow, CEP X/Y for monitoring sex-mismatched bone marrow transplants, UroVysion Multicolor Probe (bladder cancer detection), TelVysion (telomere analysis), and chromosome paints (WCP) for cultured lymphocytes. For greater versatility, the user can program custom pretreatment protocols and histological staining protocols. The end result is increased flexibility, greater consistency, reduced labor, and improved efficiency.
Materials and Methods Tissue Preparation Paraffin Sections. Multiple 5-m paraffinembedded breast tumor sections were obtained from the Department of Pathology at the Northwestern University School of Medicine (Chicago, IL). Paraffin-embedded breast tumor blocks were obtained from the Cooperative Human Tissue Net-
Fig. 1. VP 2000.
work, Midwest Division, at The Ohio State University (Columbus, OH). The tissue blocks were sliced at 5 m and mounted onto SuperFrost positively charged slides (Shandon, Inc, Pittsburgh, PA) at Vysis, Inc. Multiple slides were made from each specimen. All slides were baked at 56°C overnight and then stored at room temperature until used for pretreatment and hybridization. Urine. Uroepithelial cells were isolated from urine specimens obtained from BioQual (Plainville, MA) and from healthy donors. All samples were processed within 24 hours of collection. First, the urine was concentrated by centrifugation at 600g for 10 minutes. The pellet was resuspended in 25 to 50 mL 1X phosphate-buffered saline and concentrated at 600g for an additional 5 minutes. The pellets were resuspended slowly in 1 mL fixative (3:1, methanol and acetic acid). The specimens were concentrated at 600g for an additional 5 minutes. The fixation was repeated two more times. After the third centrifugation in fixative, the supernatant was removed, and the cell pellets were resuspended in 0.5 to 1 mL fresh fixative. Aliquots of 3 to 30 L cells from each donor were then applied to a 6-mm area in a 12-circle slide (Shandon, Inc, Rochester, MN). The slides were air-dried at ambient temperature. Other Cell Types. Bone marrow specimens were obtained from the Mayo Clinic. Uncultured primary amniocytes were obtained from Advanced BioScience Resources (Alameda, CA) and the Mayo
Automation of FISH Pretreatment
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Clinic. Cultured lymphocytes were obtained from blood samples collected from healthy donors. Bone marrow specimens, amniocytes, and cultured lymphocytes were prepared according to standard cytogenetic methods for preparing metaphase spreads [3]. For each nonparaffin specimen processed, multiple slides were prepared. Unless otherwise stated, slides were prepared up to 1 year in advance and stored at −20°C until the day of the assay. Duplicate specimen slides were pretreated manually and by the VP 2000 on the same days for comparison.
ter line with a drain tube. The water basin holds 1,650 mL water and is capable of operating in either a recirculating or captive mode. It can process up to 50 slides simultaneously. The VP 2000 is controlled by a personal computer through an RS-232 serial interface that has been programmed with a specific protocol that controls the sequence of reagents, timing, and temperatures. The slide carrier, manually mounted to a robotically controlled arm, is capable of holding up to 50 standard microscope slides.
Instrument
The following reagents for the VP 2000 are manufactured and distributed by Vysis, Inc: pretreatment solution; protease buffer; protease I and II; 2X standard saline citrate (SSC), pH 7.0; and 2 M magnesium chloride. A fixative consisting of 1% formaldehyde and 18 mm magnesium chloride in 1X phosphate-buffered saline was used on bone marrow, amniocytes, uroepithelial cells, and cultured lymphocyte Hemo-De specimens. Other reagents included Hemo-de (Fisher Scientific, Pitts-
The VP 2000 is a modification of design of the HMS/DS50 histology stainer (Carl Zeiss, Inc, Thornwood, NY), as shown in Fig. 1. Three heated basins replaced the front row of eight ambient basins that were in the HMS/DS50. The instrument has three heated reagent basins, 12 ambient reagent basins, a circulating water bath, and a drying station. The instrument is connected to a dionized wa-
Sample Pretreatment
Table 1. Comparison of VP 2000 and Manual Protocols for PathVysion (HER-2) VP 2000 Step
Description
Manual Time (min)
Step
Description
Time (min)
5 5 5 1 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Hemo-de 1 Hemo-de 2 Hemo-de 3 95%–100% Ethanol 1 95%–100% Ethanol 2 Dry on 45°C–50°C slide warmer 0.2 N HCl acid Distilled water rinse 2X SSC, pH 7, rinse Dry on 45°C–50°C slide warmer 80°C Pretreatment reagent Distilled water rinse 2X SSC, pH 7, rinse 2X SSC, pH 7, rinse 37°C Protease I 2X SSC, pH 7, rinse 2X SSC, pH 7, rinse Dry on 45°C–50°C slide warmer Neutral-buffered formalin 2X SSC, pH 7, rinse 2X SSC pH, 7, rinse Dry on 45°C–50°C slide warmer 70% Ethanol 85% Ethanol 100% Ethanol Dry on 45°C–50°C slide warmer
10 10 10 5 5 2–5 20 3 3 2–5 30 1 5 5 10 5 5 2–5 10 5 5 2–5 1 1 1
1 2 3 4 5
Hemo-de 1 Hemo-de 2 Hemo-de 3 95%–100% Ethanol 1 95%–100% Ethanol 2
6 7
0.2 N HCl Continuous-flow distilled water rinse
20 3
8 9
80°C Pretreatment reagent Continuous-flow distilled water rinse
30 3
10 11
37°C Protease I Continuous-flow distilled water rinse
10 3
12 13
Neutral-buffered formalin Continuous-flow distilled water rinse
10 3
14 15 16
70% Ethanol 85% Ethanol 100% Ethanol 25°C dry SSC, standard saline citrate.
1 1 1 3
212 Molecular Diagnosis Vol. 5 No. 3 September 2000 Table 2. Comparison of VP 2000 and Manual Protocols for Bone Marrow Pretreatment VP 2000 Step 1 2 3 4 5 6 7 8 9
Manual
Description
Time (min)
Step
Description
Time (min)
73°C 2X SSC 37°C Protease I Continuous-flow distilled water rinse 1% Formaldehyde/0.018 M MgCl2 Continuous-flow distilled water rinse 70% Ethanol 85% Ethanol 95% Ethanol 25°C dry
2 5 3 5 3 1 1 1 3
1 2 3 4 5 6 7 8 9
73°C 2X SSC 37°C Protease I 2X SSC 1% Formaldehyde/0.018 M MgCl2 2X SSC 70% Ethanol 85% Ethanol 100% Ethanol Ambient air-dry
2 5 1 5 1 1 1 1 5
SSC, standard saline citrate.
burgh, PA), neutral-buffered formalin, and 0.2 N hydrochloric acid. For assays performed using the VP 2000, 470 to 500 mL each reagent was placed in the reagent bins. For assays performed manually, 50 to 70 mL each reagent was placed in Coplin jars. Excess spaces in the slide carrier of the VP 2000 were always filled with blank slides to mimic a full-capacity run. Heated reagent basins were allowed to reach the appropriate temperature specific to the pretreatment protocol selected before the slides were loaded into the carrier and the program initiated. The slides were dried on completion of the run. The slides were removed, and FISH assays were performed. Table 1 lists the automated and manual pretreatment assays for paraffin-embedded breast tumors. Table 2 describes the automated and manual pretreatment assays for bone marrow samples. Table 3 lists the automated and manual pretreatment assays for uncultured primary amniocytes. After the cell preparations were performed,
the slides were aged overnight and stored at –20°C until use. Table 4 lists the automated and manual pretreatment assays for uroepithelial cells. Table 5 lists the automated and manual pretreatment assays for cultured lymphocytes. FISH All DNA probes and other reagents required for hybridization were manufactured at Vysis, Inc. The HYBrite denaturation hybridization instrument, manufactured by Source Scientific, is marketed and distributed exclusively by Vysis, Inc. Pretreated paraffin-embedded breast tumor sections were hybridized with PathVysion HER-2 probe (LSI HER-2 Spectrum-Orange and CEP 17 SpectrumGreen). Pretreated specimen slides were first placed on a HYBrite, set at 37°C holding temperature. The hybridization mixture was applied to the specimen slides. A glass coverslip was imme-
Table 3. Comparison of Protocols for Uncultured Amniocyte Pretreatment VP 2000 Step 1 2 3 4 5 6 7 8 9
Manual
Description
Time (min)
Step
Description
Time (min)
73°C 2X SSC incubation 37°C Protease I treatment Continuous-flow distilled water rinse 1% Formaldehyde/0.018 M MgCl2 incubation Continuous-flow distilled water rinse 70% Ethanol dehydration 85% Ethanol dehydration 95% Ethanol dehydration 25°C dry
2 5 3 5 3 1 1 1 3
1 2 3 4 5 6 7 8 9
37°C 2X SSC incubation 37°C Protease I treatment 1X PBS 1% Formaldehyde/0.018 M MgCl2 incubation 1X PBS 70% Ethanol dehydration 85% Ethanol dehydration 100% Ethanol dehydration Ambient air-dry
60 13 5 5 5 1 1 1 5
For AneuVysion, CEP probes and LSI probes are hybridized separately but on the same slide; the area of hybridization is such that there is room for two hybridizations on the same slide. SSC, standard saline citrate; PBS, phosphate-buffered saline.
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Table 4. Comparison of VP 2000 and Manual Protocols for Uroepitheliel Cells VP 2000 Step 1 2 3 4 5 6 7 8 9 10
Manual
Description
Time (min)
Step
Description
73°C 2X SSC aging Continuous-flow distilled water rinse 37°C Protease I treatment Continuous-flow distilled water rinse Postfixation Continuous-flow distilled water rinse 70% Ethanol 85% Ethanol 95% Ethanol: 5% methanol 25°C air-dry
2 3 10 3 5 3 1 1 1 3
1
73°C 2X SSC aging
2 3 4 5 6 7 8 9
37°C Protease I treatment 1X PBS rinse Postfixation 1X PBS rinse 70% Ethanol 85% Ethanol 100% Ethanol Air-dry
Time (min) 2 10 5 5 5 1 1 1 3
SSC, standard saline citrate; PBS, phosphate-buffered saline.
diately applied, and the edges of the coverslip were sealed with rubber cement. The specimens were then codenatured at 73°C for 5 minutes and hybridized at 37°C for 16 to 18 hours. After hybridization, the slides were removed from the HYBrite and the rubber cement was carefully removed from the slides. The slides were soaked in room temperature 2X SSC/0.3% Nonidet P40 (Sigma-Aldrich, St. Louis, MO) at ambient temperature for 2 to 10 minutes to remove the coverslips. The slides were then immersed in 73°C 2X SSC/0.3% Nonidet 40 for 2 minutes to remove the nonspecifically bound probe, then air dried in the dark. DAPI I counterstain (Vysis, Inc) was applied to the specimen to allow visualization of the nucleus. Visualization and scoring was performed using DAPI/green/orange triple bandpass, DAPI/green dual pass, and DAPI/orange dual pass filter sets. Pretreated bone marrow samples were hybridized with CEP 8 (SpectrumOrange), CEP 12 (SpectrumOrange), CEP X/Y (CEP × SpectrumOrange and
CEP Y SpectrumGreen), and bcr/abl and bcr/abl ES (LSI bcr SpectrumGreen and LSI abl SpectrumOrange) hybridization mixtures. For the CEP probes, the slides were first placed on a 42°C HYBrite, and the probe hybridization mixture was applied to each slide. Coverslips were applied and sealed as previously described. The slides were codenatured at 75°C for 1 minute and hybridized at 42°C for 1 hour. For the bcr/abl mixes, the slides were placed on a 37°C HYBrite, and the probe hybridization mixture was applied to each slide. Coverslips were applied and sealed as previously described. The specimen and probe were codenatured at 73°C for 5 minutes and hybridized at 37°C for 17 hours. Pretreated uncultured primary amniocytes were hybridized with the AneuVysion Assay. AneuVysion consists of two mixes that are hybridized on the same slide but under different coverslips. The first mix contains CEP Y SpectrumOrange, CEP X Spectrum-Green, and CEP 18 SpectrumAqua. The
Table 5. Comparison of VP 2000 and Manual Protocols for Cultured Lymphocyte Pretreatment VP 2000 Step 1 2 3 4 5 6 7 8 9
Manual
Description
Time (min)
Step
Description
Time (min)
37°C 2X SSC incubation 1% Formaldehyde/0.018 M MgCl2 incubation Continuous-flow distilled water rinse 37°C Protease II incubation Continuous-flow distilled water rinse 70% Ethanol dehydration 85% Ethanol dehydration 95% Ethanol dehydration 25°C dry
10 15 3 13 3 1 1 1 3
1 2 3 4 5 6 7 8 9
37°C 2X SSC incubation 1% Formaldehyde/0.018 M MgCl2 incubation 2X SSC 37°C Protease II incubation 2X SSC 70% Ethanol dehydration 85% Ethanol dehydration 100% Ethanol dehydration Ambient air-dry
10 15 5 13 5 1 1 1 5
SSC, standard saline citrate.
214 Molecular Diagnosis Vol. 5 No. 3 September 2000
second mix contains LSI 21 SpectrumOrange and LSI 13 SpectrumGreen. The slides were placed on a 37°C HYBrite, and 10 L each hybridization mixture was applied to each slide. The slides were codenatured at 73°C for 5 minutes and hybridized at 37°C for 6 hours. Pretreated uroepithelial cells were hybridized with UroVision Multicolor Probe, which contains CEP 3 SpectrumRed, CEP 7 SpectrumGreen, CEP 17 SpectrumAqua, and LSI 9p21 SpectrumGold™. The 12-circle slides were placed on a 39°C HYBrite, and 3 L hybridization mixture was applied to each circle that contained a specimen. Coverslips were applied and sealed as previously described. There were two to four circles with hybridization buffer underneath each coverslip. The specimen and probe were codenatured at 73°C for 2 minutes. Hybridization was at 39°C for 4 hours. Pretreated cultured lymphocytes were hybridized with TelVysion 17p SpectrumGreen, TelVysion 2p SpectrumGreen, TelVysion 3q SpectrumOrange,
TelVysion 21q SpectrumOrange, WCP 3 SpectrumGreen, and WCP 3 SpectrumOrange. The slides were placed on a 37°C HYBrite, and 10 L each hybridization mixture was applied to each slide. Coverslips were applied and sealed as previously described. The specimen and probes were codenatured at 70°C for 3 minutes. Telomere probes were hybridized at 37°C for 6 hours, and WCP probes were hybridized at 37°C for 16 hours. To remove nonspecifically bound probe, all specimen types were washed according to the manufacturer’s product insert. Specifically, after the slides were removed from the HYBrite, the coverslips were removed, and the slides were immediately placed in 73°C ± 1°C 0.4× SSC/0.3% Nonidet 40 in Coplin jars for 2 minutes. They were then placed in room temperature 2X SSC/0.1% Nonidet 40 for 1 minute, dried in the dark, and counterstained with DAPI II (Vysis, Inc, Downers Grove, IL). Visualization and scoring were performed us-
Fig. 2. Scoring criteria for FISH. (A) Example of a score of 2 on paraffin-embedded breast cancer section hybridized with FISH probes. For a passing score of 3, there should be clear, distinct signals in at least 80% of the cells. Nonspecific hybridization should be light enough and uniform enough to allow visualization of the signals. Here, less than 80% of the cells have distinct signals. Nonspecific hybridization is somewhat particulate and heavy enough to interfere with easy visualization of signals. (B) Example of a score of 4 on paraffin-embedded breast cancer section hybridized with FISH probes. For a score of 4, there should be clear, distinct signals in at least 90% of the cells. Nonspecific hybridization should be light enough and uniform enough to allow for easy visualization of the signals. Here, all cells in the same plane of focus have clear, distinct signals. There is light nonspecific hybridization in the background, but it does not interfere with visualization of the signals.
Automation of FISH Pretreatment
ing the manufacturer’s suggested filter sets appropriate for each test. Slide Evaluation Slides were rated on a scale of 1 to 5 for quality of hybridization, with 5 the highest possible score. A slide must have a score of 3 or greater for all evaluated parameters to be of acceptable quality. A score of 5 corresponds to exceptional quality, 4 corresponds to high quality, 3 corresponds to a slide that can be enumerated with reasonable ease, 2 corresponds to a slide in which signal is visible but not easily enumerated, and 1 corresponds to hybridization failure. Examples of a score of 4 and 2 are
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Jacobson et al. 215
shown in Fig. 2. Parameters evaluated were signal intensity, signal specificity, general background, cross-hybridization, and overall quality. Specimen loss was also evaluated for paraffin-embedded breast tissue sections. A score of 5 corresponded to no tissue loss; 4, less than 10% loss; 3, 11% to 25% loss; 2, 26% to 75% loss; and 1, greater than 75% loss. Greater than 10% tissue loss was seen on only a few specimens. Two raters independently evaluated each slide, and slides were blinded to type of pretreatment. The scores were analyzed using JMP (SAS Institute, Inc, Cary, NC) statistical software program and analyzed for significance using Student’s t-test. P less than .05 and R2 greater than 0.81 is considered statistically significant.
Table 6. Manual versus Automation Using the VP 2000: Specimen Pretreatment of Paraffin Section Slide Before Vysis FISH Manual Method
Incubation Time (min)
1. Reagent preparation and start-up Reagent prep 30 Load slides 1 Subtotal 2. Deparaffinization Hemo-De Hemo-De Hemo-De Ethanol Ethanol Subtotal 3. Pretreatment 0.2 N HCl Purified H2O Wash 2X SSC Pretreatment Purified H2O Wash 2X SSC Wash 2X SSC Subtotal 4. Enzyme digestion Protease Wash 2X SSC Wash 2X SSC Subtotal 5. Fixation 10% Formalin Wash 2X SSC Wash 2X SSC Subtotal 6. Dry slide Subtotal Total
31 10 10 10 5 5 40 20 3 3 30 1 5 5 67 10 5 5 20
Captive Time (min) 30 31
45
27
25
10 5 5 20 5 5 183
SSC, standard saline citrate.
25 1 0 154
VP 2000 Automation 1. Reagent preparation and start-up Reagent prep VP 2000 Processor start up Load slides Subtotal 2. Deparaffinization Hemo-De Hemo-De Hemo-De EtOH EtOH Subtotal 3. Pretreatment 0.2 N HCl Water rinse Pretreatment reagent Water rinse
Incubation Time (min)
Captive Time (min)
10 1 3 14
10 1 14
5 5 5 1 1 17
0
20 3 30 3
Subtotal 4. Enzyme digestion Protease I solution Water rinse
56
0
Subtotal 5. Fixation 10% Formalin Water rinse 70% ETOH 85% ETOH 100% ETOH Subtotal 6. Dry slide Subtotal
13
0
10 3 1 1 1 16 3 3 119
0 0 0 14
10 3
216 Molecular Diagnosis Vol. 5 No. 3 September 2000
Results Time Savings A comparison of the individual protocols for the VP 2000 and manual format is listed in Tables 1 through 5. As well as the time savings afforded by the walk-away feature of the automated protocol, the actual time to completion is significantly shortened in some protocols with automation. The optimal VP 2000 protocol was developed through empirical validation on different sample types. The protocol modifications in the VP 2000 were made for two primary reasons: total run time and reagent stability. In the automated protocol, many steps were shortened because an equivalent level of performance was achieved. This was because of the greater ratio of reagent to sample, resulting in a more effective dilution or treatment. Typically, a Coplin jar containing 50 mL reagent could process up to six slides, whereas the VP 2000 basins hold approximately 650 mL, for up to 50 slides. The second reason for the modifications is reagent stability. There is significant carry over of reagents with a fully loaded slide carrier. Although all reagents have been optimized with respect to concentration and pH, certain reagents, such as the 0.2 N HCl, pretreatment reagent, and proteases, are particularly sensitive to dilution effects and pH changes from the preceding reagent, which is typically 2X SSC. Prior rinsing of the sample slides in deionized water resulted in a significant improvement in reagent stability. In the case of amniocytes, manual 2X SSC pretreatment step no. 1 was kept at 37°C for 60 minutes because it follows the stated protocol in the package insert. However, manual
pretreatment at 73°C for 2 minutes works equivalently. A significant advantage of the VP 2000 Processor is the reduction in hands-on labor that it potentially provides to the laboratory. By reducing handson labor requirements in specimen pretreatment procedures, the technologist is freed to perform other tasks, providing greater efficiency and use of resources. This may allow resource-constrained laboratories a feasible solution for adopting FISH assays into their laboratories As described previously, certain tissue specimens must undergo a pretreatment process before performing the FISH assay. One particular specimen type that requires pretreatment is sectioned paraffin-embedded tissue. To prepare this type of specimen for the FISH assay, the specimen must undergo deparaffinization to dissolve the paraffin surrounding the tissue, followed by an enzymatic digestion to minimize proteins and connective tissue in and surrounding the cells. This procedure consists of 18 manual steps and takes approximately 183 minutes to complete. Manual processing using this procedure requires movement of the slides through a variety of reagents, incubating the slides in the reagents for specified periods of time, and then moving them to the next reagent. The incubation times associated with each step of the process range from 1 to 30 minutes, often leaving little free time for the technologist to perform other tasks within the laboratory while using this process. Use of the VP 2000 for the pretreatment process essentially eliminates all hands-on processing, thus freeing the technologist to perform other analyses or tasks. A comparative analysis of processing and incubation time and labor costs for the manual method
Table 7. Hands-on Time Savings Per Slide From VP 2000 Pretreatment VP 2000
Sample Type Uncultured amniocytes Bone marrow WCPs/TelVysion Uroepitheliel cells
Manual
Run Time (min)
Hands-on Time (min)
Total Hands-on Time/Slide (min)
Run Time (min)
Hands-on Time (min)
Total Hands-on Time/Slide (min)
Reduction in Processing Time/Slide (%)
24 24 47 32
30 30 30 30
0.6 0.6 0.6 0.6
96 + 30* 22 + 30* 56 + 30* 33 + 30*
146 72 106 83
2.9 1.4 2.1 1.7
79 57 71 65
Assay time is based on a batch size of 50 slides. For the VP 2000 protocol, hands-on time includes reagent preparation, loading slide rack, and selecting and starting a program. For the manual assay, hands-on time includes reagent preparation, incubation time, and slide handling during run. *Time required for slide handling.
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Jacobson et al. 217
Table 8. Results for PathVysion (HER-2) Mean Score Attribute
Range
VP 2000
Manual
VP 2000
Manual
Significance
3.88 3.91 4.11 4.20 4.78
3.27 3.39 3.83 4.19 4.37
3.00–4.25 3.25–4.25 3.75–4.25 3.50–4.35 4.00–5.00
1.75–4.00 2.00–4.13 2.75–4.50 3.50–4.50 1.50–5.00
.0000 .0000 .0000 NS .0000
Overall readability Probe intensity Probe specificity Background Tissue loss
All specimens were run pairwise. N ⳱ 16. NS, not significant.
versus the VP 2000 automated method for pretreatment procedure is listed in Table 6 . This analysis provides an estimated time comparison based on experience with both manual and automated methods as performed at Vysis, Inc. All time data points are recorded in minutes. For purposes of the analysis, the procedure was divided into six major sections: (1) reagent preparation and start-up, (2) deparaffinization, (3) pretreatment, (4) enzyme digestion, (5) fixation, and (6) drying. Within each section, the reagent used in the proFig. 3. Comparison of hybridization quality using manual and VP 2000 pretreatment protocols. (A) HER-2–amplified paraffin-embedded breast tumor section processed with manual pretreatment before hybridization. HER-2 probe is orange, and CEP 17 is green. Nuclear staining is blue (DAPI). Larger nuclei with multiple HER-2 signals are malignant, and smaller cells with one to two copies of each probe are normal. Some nuclei have been truncated by sectioning and thus have fewer than two copies of each probe. Signals can be enumerated, but not easily, as for the section on the right that was processed on the VP 2000. (B) HER-2–amplified paraffin-embedded breast tumor section from the same patient processed with VP 2000 pretreatment before hybridization. HER-2 probe is orange, and CEP 17 is green. Nuclear staining is blue (DAPI). Visualization of fluorophores is easier because more efficient digestion reduces autofluorescence of extracellular elements.
cedural step, incubation time (time that the slide remains in the reagent), and captive time (time that the technologist is practically required to perform the task or wait for the task to be completed) is described. Each procedure section is then subtotaled to provide information on time expended for that particular section of the process. The captive time for each step represents the true labor associated with the process. In cases in which the incubation time is short (ⱕ10 minutes), it is assumed that it would not be practical for the technologist to aban-
218 Molecular Diagnosis Vol. 5 No. 3 September 2000 Table 9. Results for AneuVysion Mean Scores Probe CEP 18, X, and Y LSI 13 and 21
Range
VP 2000
Manual
VP 2000
Manual
Significance
4.19 3.50
3.94 3.44
4.00–4.50 3.25–3.75
3.50–4.25 3.00–3.75
NS NS
All specimens were run pairwise. N ⳱ 8. NS, not significant.
don the process to perform other laboratory tasks; therefore, they are committed to the entire time span for that step. In some procedures, the steps of the manual method do not match the VP 2000 Processor method because the procedures are different. The total time for each method represents the time in minutes for the entire manual or VP 2000 process, incubation, and captive or labor time. The manual method for this procedure results in an incubation time of 183 minutes and a captive time of 154 minutes. The VP 2000 Processor automated procedure results in an incubation time of 119 minutes and captive time of only 14 minutes. A theoretical calculation of labor costs for this analysis based on an average hourly labor cost (including salary wage and benefits) of $40 results in a total labor cost of $102.66 for the manual procedure method and $9.33 for the VP 2000 method. Considering that the maximum number of slides that one would realistically be able to process per batch using the manual method is 10 slides, labor would cost $10.22 per slide in this scenario. In contrast, the VP 2000 Processor has the capacity to process 50 slides per batch, resulting in an extremely reduced labor cost of $0.19 per slide, which provides a significant cost savings to the laboratory. A summary of the time savings for other assays is listed in Table 7. Specifically, for a batch of 50 slides, the time savings ranged from 65% for uroepithelial
cells, 71% for lymphocytes, and 79% for amniocytes. Table 8 lists the results of pretreatment and hybridization on paraffin-embedded breast tissue sections using PathVysion HER-2. VP 2000 pretreatment resulted in statistically better results than the manual pretreatment protocol. This assay showed the greatest overall difference in performance between the VP 2000 and the manual format. Probe signal intensity and specificity were greater with the VP 2000 pretreatment than with the manual assay (Fig. 3). No tissue loss greater than 10% was seen with VP 2000 pretreatment. Conversely, a few samples (three of 16 samples) showed extensive tissue loss with the manual protocol. Mistakes during handling, such as placing a slide in the wrong Coplin jar or scraping a specimen, are essentially eliminated using the VP 2000 Processor. Replacement of 2X SSC solution rinses with continuousflow water rinses may also have influenced the outcome. The continuous-flow water rinse allows for more complete washing of the specimen and eliminates contamination of reagents with 2X SSC. Table 9 lists the results of pretreatment and hybridization on uncultured primary amniocytes with AneuVysion. In this case, the results of VP 2000 pretreatment and manual pretreatment were equivalent. This is consistent with the lower complexity of the pretreatment assay compared with PathVysion
Table 10. Results for Bone Marrows Mean Scores Probe CEP 8 CEP 12 CEP X/Y LSI bcr/abl
Range
VP 2000
Manual
VP 2000
Manual
Significance
3.81 3.37 3.81 2.88
3.44 2.81 3.69 3.13
3.00–4.50 2.75–4.50 3.50–4.25 2.50–3.25
3.00–4.00 2.50–3.25 3.25–4.50 2.75–3.50
.0340 NS NS NS
All specimens were run pairwise. N ⳱ 4 for each probe set. NS, not significant.
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Table 11. Results for PathVysion Bladder Probe SpectrumGold LSI 9p21 SpectrumAqua CEP 17 SpectrumGreen CEP 7 SpectrumRed CEP 3
VP 2000 Mean Scores
Manual Mean Scores
Significance
3.47 4.48 4.50 4.76
3.27 4.42 4.40 4.71
.0051 .4139 .3219 .5644
All specimens were run pairwise. N ⳱ 15.
(HER-2). Table 10 lists the results of pretreatment and hybridization on bone marrow cells. CEP 8 was improved by VP 2000 treatment, and there was no difference between manual and VP 2000 pretreatment for CEP 12, CEP X/Y, and LSI bcr/abl. Again, the smaller differences between VP 2000 and manual assays are consistent with the relatively low complexity of this pretreatment. Table 11 lists the results of pretreatment and hybridization on uroepithelial cells with UroVysion. There were no differences with CEP 3, CEP 7, and CEP 17, but signal quality for LSI 9p21 was slightly better with the VP 2000 pretreatment. Table 12 lists the results of pretreatment and hybridization on cultured lymphocyte metaphase spreads with telomere probes and Chromosome Paints. WCP 3 SpectrumOrange and SpectrumGreen performed the same regardless of assay. TelVysion probes, 2p SpectrumGreen, 17p SpectrumGreen, and 21q SpectrumOrange were statistically significantly improved by the VP 2000 assay. Average scores for 3q SpectrumOrange were greater with the VP 2000 assay, but the difference was not statistically significant.
Discussion A variety of cell pretreatment protocols have been known to improve FISH results for the detec-
tion of chromosome copy number aberration. Formalin-fixed, paraffin-embedded tissues are particularly refractory to FISH hybridization [4,5]. The number of individual manipulations required to manually process large numbers of slides containing this sample type is demanding and can result in uneven pretreatment of specimens in the hands of inexperienced users. Slight variations in individual reagent exposures and manual manipulation of slides can result in a large cumulative variation in pretreatment efficacy. We have shown that automation of all pretreatment steps greatly enhances the performance of FISH for PathVysion HER-2, as well as for a variety of FISH probes. This is particularly true on marginal samples, which were deliberately included in this study to reflect the wide variation in sample quality. Some of these variables can be controlled in the hands of experienced users, such as the preparation of metaphase slides or lymphocytes. When specimens are prepared under optimal conditions, FISH performance is optimal [6,7]. However, in the case of archived specimens, such as paraffin-embedded formalin-fixed tissue, optimization of pretreatment is one of the few options available. This study clearly shows that the greatest benefit of automated pretreatment is seen with these difficult samples. This outcome suggests that difficult samples are not refractory to standard
Table 12. Results for TelVysion and WCPs Mean Scores Probe WCP 3, SpectrumOrange WCP 3, SpectrumGreen 2p, SpectrumGreen telomere 3q, SpectrumOrange telomere 17p, SpectrumGreen telomere 21q, SpectrumOrange telomere
Range
VP 2000
Manual
VP 2000
Manual
Significance
3.50 3.33 3.81 3.25 3.62 3.38
3.50 3.33 1.69 2.25 1.38 2.19
3.50 3.25–3.50 3.75–4.00 2.75–3.75 3.50–3.75 3.00–3.75
3.25–4.00 3.25–3.50 1.25–2.00 1.75–2.50 1.00–2.25 1.50–2.50
NS NS .000 NS .0000 .0000
All specimens were run pairwise. For the WCP probes, n ⳱ 3. For the telomere probes, n ⳱ 4. NS, not significant.
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pretreatment regimens but are exquisitely sensitive to overtreatment or undertreatment. This hypothesis could explain our observations on the variability in assay performance of the manual format on marginal samples. In replicate adjacent sections from the tissue block, the manual format shows much greater variations between runs than the VP 2000. The other major benefits for automation of pretreatment are the significant reductions in labor and time to result. These savings can also be realized in the development of custom protocols for special applications. The movement of FISH technology toward full automation is likely to achieve the same benefits observed with immunohistochemistry assays in which artistry is removed and improvements in quality, speed, and reproducibility are necessary for improved adoption of the technology in the clinical laboratory [8]. Additionally, the achievement of this level of quality for FISH can provide a greater level of sensitivity and specificity with less ambiguity than other competing technologies. Received February 24, 2000. Received in revised form March 14, 2000. Accepted May 22, 2000.
2.
3.
4.
5.
6.
7.
References 1. Hopman AHN, Poddighe P, Moesker O, Ramaekers FCS: Interphase cytogenetics: An approach to the
8.
detection of genetic aberrations in tumors. In McGee JO, Herrington CS: Diagnostic molecular pathology: A practical approach. New York, NY, IRL Press, 1991, pp. 141–167 Hopman AH, Voorter CE, Ramaekers FC: Detection of genomic changes in cancer by in situ hybridization. Mol Biol Rep 1994;19:31–44 Pinkel D, Straume T, Gray JW: Cytogenetic analysis using quantitative, high sensitivity fluorescence hybridization. Proc Natl Acad Sci U S A 1986;83: 2934–2938 Hyytinen E, Visakorpi T, Kallioniemi A, Kallioniemi OP, Isola JJ: Improved technique for analysis of formalin-fixed, paraffin-embedded tumors by fluorescence in situ hybridization. Cytometry 1994;16: 93–99 Kapranos N, Kontogergos G, Frangia K, Kokka E: Effect of fixation on interphase cytogenetic analysis by direct fluorescence in situ hybridization on cell imprints. Biotechnol Histochem 1997;72:148– 151 Persons DL, Bui MM, Lowery MC, et al.: Fluorescence in situ hybridization (FISH) for detection of Her-2/neu amplification in breast cancer: A multicenter portability study. Ann Clin Lab Sci 2000;1: 41–48 Abati A, Sanford JS, Fetsch P, Marincola FM, Wolman SR: Fluorescence in situ hybridization (FISH): A user’s guide to optimal preparation of cytologic specimens. Diagn Cytopathol 1995;13:486–492 Grogan TM: Automated immunohistochemical analysis. Am J Clin Pathol 1992;98(Suppl 1):S35-S38
Automation of Fluorescence In Situ Hybridization Pretreatment: A Comparative Study of Different Sample Types KRISTINE JACOBSON, MS, ANTHONY THOMPSON, MA, GERRY BROWNE, BS, CHRISTINA SHASSERRE, MS, STEVEN A. SEELIG, MD, PhD, WALTER KING, PhD Downers Grove, Illinois
Background: In fluorescence in situ hybridization (FISH) applications, the efficiency of probe hybridization is greatly enhanced by treating the cell or tissue preparation with a variety of reagents that make the target permeable while preserving important morphological features. Pretreatment protocols can be very labor intensive, adding cost to the test. The automation of specimen pretreatment eliminates human variation in the procedure through standardization of such variables as treatment times and temperatures. Methods and Results: We developed an instrument, the VP 2000 Processor, that can process up to 50 specimens simultaneously under computer control. In this comparative FISH study of matched specimens, one set was processed according to the manual pretreatment protocol and compared with specimens processed by the VP 2000. Processed specimen types included paraffin-embedded breast tissue, uncultured amniocytes, bone marrow, peripheral-blood lymphocytes, and uroepithelial cells recovered from urine. Data show equivalent or brighter and more specific overall signal quality compared with matched manually executed controls. Conclusions: The automation of sample pretreatment for FISH provides a superior, more consistent level of performance than the manual format. Key words: fluorescence in situ hybridization, centromeric enumeration probes, locus-specific identifier probes, chromosome painting probes.
before FISH. The amount of labor required varies with the type of specimen, but it can be extensive with certain specimen types, such as paraffinembedded tissue [1,2]. We have developed an instrument, the VP 2000 Processor (Vysis, Inc, Downers Grove, IL), that automates a variety of pretreatment protocols on 50 specimen slides at the same time. Together with the HYBrite (Vysis, Downers Grove, IL), which automates denaturation and hybridization, hands-on processing time for directly labeled FISH probe assays is reduced 57% to 90%. Pretreatment protocols for paraffin-embedded tumor sections, uncultured amniocytes, bone marrow, uroepithelial
Fluorescent in situ hybridization (FISH) technology is becoming more widely used in today’s pathology laboratory. Despite the performance advantages in specificity and sensitivity, FISH has remained in a manual-assay format. A significant fraction of the labor required for optimal FISH performance involves the pretreatment of specimens From Vysis, Inc, Downers Grove, IL. Reprint requests: Walter King, PhD, Director, Assay Development, Vysis, Inc, 3100 Woodcreek Dr, Downers Grove, IL 60515. Email: [email protected] Copyright © 2000 by Churchill Livingstone威 1084-8592/00/0503-0007$10.00/0 doi:10.1053/modi.2000.9731
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cells, and cultured lymphocytes are preprogrammed into the instrument software. The instrument has been validated in pretreatment protocols for several specific FISH assay specimen types: Vysis PathVysion (Vysis, Inc, Downers Grove, IL) (c-erb B2 [HER-2] and chromosome 17 enumeration probe [CEP 17] on paraffin-embedded breast tumor sections), the AneuVysion Assay (test for common prenatal aneuplodies on uncultured amniocytes), CEP 8, CEP 12, and LSI bcr/abl for leukemia diagnosis on bone marrow, CEP X/Y for monitoring sex-mismatched bone marrow transplants, UroVysion Multicolor Probe (bladder cancer detection), TelVysion (telomere analysis), and chromosome paints (WCP) for cultured lymphocytes. For greater versatility, the user can program custom pretreatment protocols and histological staining protocols. The end result is increased flexibility, greater consistency, reduced labor, and improved efficiency.
Materials and Methods Tissue Preparation Paraffin Sections. Multiple 5-m paraffinembedded breast tumor sections were obtained from the Department of Pathology at the Northwestern University School of Medicine (Chicago, IL). Paraffin-embedded breast tumor blocks were obtained from the Cooperative Human Tissue Net-
Fig. 1. VP 2000.
work, Midwest Division, at The Ohio State University (Columbus, OH). The tissue blocks were sliced at 5 m and mounted onto SuperFrost positively charged slides (Shandon, Inc, Pittsburgh, PA) at Vysis, Inc. Multiple slides were made from each specimen. All slides were baked at 56°C overnight and then stored at room temperature until used for pretreatment and hybridization. Urine. Uroepithelial cells were isolated from urine specimens obtained from BioQual (Plainville, MA) and from healthy donors. All samples were processed within 24 hours of collection. First, the urine was concentrated by centrifugation at 600g for 10 minutes. The pellet was resuspended in 25 to 50 mL 1X phosphate-buffered saline and concentrated at 600g for an additional 5 minutes. The pellets were resuspended slowly in 1 mL fixative (3:1, methanol and acetic acid). The specimens were concentrated at 600g for an additional 5 minutes. The fixation was repeated two more times. After the third centrifugation in fixative, the supernatant was removed, and the cell pellets were resuspended in 0.5 to 1 mL fresh fixative. Aliquots of 3 to 30 L cells from each donor were then applied to a 6-mm area in a 12-circle slide (Shandon, Inc, Rochester, MN). The slides were air-dried at ambient temperature. Other Cell Types. Bone marrow specimens were obtained from the Mayo Clinic. Uncultured primary amniocytes were obtained from Advanced BioScience Resources (Alameda, CA) and the Mayo
Automation of FISH Pretreatment
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Jacobson et al. 211
Clinic. Cultured lymphocytes were obtained from blood samples collected from healthy donors. Bone marrow specimens, amniocytes, and cultured lymphocytes were prepared according to standard cytogenetic methods for preparing metaphase spreads [3]. For each nonparaffin specimen processed, multiple slides were prepared. Unless otherwise stated, slides were prepared up to 1 year in advance and stored at −20°C until the day of the assay. Duplicate specimen slides were pretreated manually and by the VP 2000 on the same days for comparison.
ter line with a drain tube. The water basin holds 1,650 mL water and is capable of operating in either a recirculating or captive mode. It can process up to 50 slides simultaneously. The VP 2000 is controlled by a personal computer through an RS-232 serial interface that has been programmed with a specific protocol that controls the sequence of reagents, timing, and temperatures. The slide carrier, manually mounted to a robotically controlled arm, is capable of holding up to 50 standard microscope slides.
Instrument
The following reagents for the VP 2000 are manufactured and distributed by Vysis, Inc: pretreatment solution; protease buffer; protease I and II; 2X standard saline citrate (SSC), pH 7.0; and 2 M magnesium chloride. A fixative consisting of 1% formaldehyde and 18 mm magnesium chloride in 1X phosphate-buffered saline was used on bone marrow, amniocytes, uroepithelial cells, and cultured lymphocyte Hemo-De specimens. Other reagents included Hemo-de (Fisher Scientific, Pitts-
The VP 2000 is a modification of design of the HMS/DS50 histology stainer (Carl Zeiss, Inc, Thornwood, NY), as shown in Fig. 1. Three heated basins replaced the front row of eight ambient basins that were in the HMS/DS50. The instrument has three heated reagent basins, 12 ambient reagent basins, a circulating water bath, and a drying station. The instrument is connected to a dionized wa-
Sample Pretreatment
Table 1. Comparison of VP 2000 and Manual Protocols for PathVysion (HER-2) VP 2000 Step
Description
Manual Time (min)
Step
Description
Time (min)
5 5 5 1 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Hemo-de 1 Hemo-de 2 Hemo-de 3 95%–100% Ethanol 1 95%–100% Ethanol 2 Dry on 45°C–50°C slide warmer 0.2 N HCl acid Distilled water rinse 2X SSC, pH 7, rinse Dry on 45°C–50°C slide warmer 80°C Pretreatment reagent Distilled water rinse 2X SSC, pH 7, rinse 2X SSC, pH 7, rinse 37°C Protease I 2X SSC, pH 7, rinse 2X SSC, pH 7, rinse Dry on 45°C–50°C slide warmer Neutral-buffered formalin 2X SSC, pH 7, rinse 2X SSC pH, 7, rinse Dry on 45°C–50°C slide warmer 70% Ethanol 85% Ethanol 100% Ethanol Dry on 45°C–50°C slide warmer
10 10 10 5 5 2–5 20 3 3 2–5 30 1 5 5 10 5 5 2–5 10 5 5 2–5 1 1 1
1 2 3 4 5
Hemo-de 1 Hemo-de 2 Hemo-de 3 95%–100% Ethanol 1 95%–100% Ethanol 2
6 7
0.2 N HCl Continuous-flow distilled water rinse
20 3
8 9
80°C Pretreatment reagent Continuous-flow distilled water rinse
30 3
10 11
37°C Protease I Continuous-flow distilled water rinse
10 3
12 13
Neutral-buffered formalin Continuous-flow distilled water rinse
10 3
14 15 16
70% Ethanol 85% Ethanol 100% Ethanol 25°C dry SSC, standard saline citrate.
1 1 1 3
212 Molecular Diagnosis Vol. 5 No. 3 September 2000 Table 2. Comparison of VP 2000 and Manual Protocols for Bone Marrow Pretreatment VP 2000 Step 1 2 3 4 5 6 7 8 9
Manual
Description
Time (min)
Step
Description
Time (min)
73°C 2X SSC 37°C Protease I Continuous-flow distilled water rinse 1% Formaldehyde/0.018 M MgCl2 Continuous-flow distilled water rinse 70% Ethanol 85% Ethanol 95% Ethanol 25°C dry
2 5 3 5 3 1 1 1 3
1 2 3 4 5 6 7 8 9
73°C 2X SSC 37°C Protease I 2X SSC 1% Formaldehyde/0.018 M MgCl2 2X SSC 70% Ethanol 85% Ethanol 100% Ethanol Ambient air-dry
2 5 1 5 1 1 1 1 5
SSC, standard saline citrate.
burgh, PA), neutral-buffered formalin, and 0.2 N hydrochloric acid. For assays performed using the VP 2000, 470 to 500 mL each reagent was placed in the reagent bins. For assays performed manually, 50 to 70 mL each reagent was placed in Coplin jars. Excess spaces in the slide carrier of the VP 2000 were always filled with blank slides to mimic a full-capacity run. Heated reagent basins were allowed to reach the appropriate temperature specific to the pretreatment protocol selected before the slides were loaded into the carrier and the program initiated. The slides were dried on completion of the run. The slides were removed, and FISH assays were performed. Table 1 lists the automated and manual pretreatment assays for paraffin-embedded breast tumors. Table 2 describes the automated and manual pretreatment assays for bone marrow samples. Table 3 lists the automated and manual pretreatment assays for uncultured primary amniocytes. After the cell preparations were performed,
the slides were aged overnight and stored at –20°C until use. Table 4 lists the automated and manual pretreatment assays for uroepithelial cells. Table 5 lists the automated and manual pretreatment assays for cultured lymphocytes. FISH All DNA probes and other reagents required for hybridization were manufactured at Vysis, Inc. The HYBrite denaturation hybridization instrument, manufactured by Source Scientific, is marketed and distributed exclusively by Vysis, Inc. Pretreated paraffin-embedded breast tumor sections were hybridized with PathVysion HER-2 probe (LSI HER-2 Spectrum-Orange and CEP 17 SpectrumGreen). Pretreated specimen slides were first placed on a HYBrite, set at 37°C holding temperature. The hybridization mixture was applied to the specimen slides. A glass coverslip was imme-
Table 3. Comparison of Protocols for Uncultured Amniocyte Pretreatment VP 2000 Step 1 2 3 4 5 6 7 8 9
Manual
Description
Time (min)
Step
Description
Time (min)
73°C 2X SSC incubation 37°C Protease I treatment Continuous-flow distilled water rinse 1% Formaldehyde/0.018 M MgCl2 incubation Continuous-flow distilled water rinse 70% Ethanol dehydration 85% Ethanol dehydration 95% Ethanol dehydration 25°C dry
2 5 3 5 3 1 1 1 3
1 2 3 4 5 6 7 8 9
37°C 2X SSC incubation 37°C Protease I treatment 1X PBS 1% Formaldehyde/0.018 M MgCl2 incubation 1X PBS 70% Ethanol dehydration 85% Ethanol dehydration 100% Ethanol dehydration Ambient air-dry
60 13 5 5 5 1 1 1 5
For AneuVysion, CEP probes and LSI probes are hybridized separately but on the same slide; the area of hybridization is such that there is room for two hybridizations on the same slide. SSC, standard saline citrate; PBS, phosphate-buffered saline.
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Jacobson et al. 213
Table 4. Comparison of VP 2000 and Manual Protocols for Uroepitheliel Cells VP 2000 Step 1 2 3 4 5 6 7 8 9 10
Manual
Description
Time (min)
Step
Description
73°C 2X SSC aging Continuous-flow distilled water rinse 37°C Protease I treatment Continuous-flow distilled water rinse Postfixation Continuous-flow distilled water rinse 70% Ethanol 85% Ethanol 95% Ethanol: 5% methanol 25°C air-dry
2 3 10 3 5 3 1 1 1 3
1
73°C 2X SSC aging
2 3 4 5 6 7 8 9
37°C Protease I treatment 1X PBS rinse Postfixation 1X PBS rinse 70% Ethanol 85% Ethanol 100% Ethanol Air-dry
Time (min) 2 10 5 5 5 1 1 1 3
SSC, standard saline citrate; PBS, phosphate-buffered saline.
diately applied, and the edges of the coverslip were sealed with rubber cement. The specimens were then codenatured at 73°C for 5 minutes and hybridized at 37°C for 16 to 18 hours. After hybridization, the slides were removed from the HYBrite and the rubber cement was carefully removed from the slides. The slides were soaked in room temperature 2X SSC/0.3% Nonidet P40 (Sigma-Aldrich, St. Louis, MO) at ambient temperature for 2 to 10 minutes to remove the coverslips. The slides were then immersed in 73°C 2X SSC/0.3% Nonidet 40 for 2 minutes to remove the nonspecifically bound probe, then air dried in the dark. DAPI I counterstain (Vysis, Inc) was applied to the specimen to allow visualization of the nucleus. Visualization and scoring was performed using DAPI/green/orange triple bandpass, DAPI/green dual pass, and DAPI/orange dual pass filter sets. Pretreated bone marrow samples were hybridized with CEP 8 (SpectrumOrange), CEP 12 (SpectrumOrange), CEP X/Y (CEP × SpectrumOrange and
CEP Y SpectrumGreen), and bcr/abl and bcr/abl ES (LSI bcr SpectrumGreen and LSI abl SpectrumOrange) hybridization mixtures. For the CEP probes, the slides were first placed on a 42°C HYBrite, and the probe hybridization mixture was applied to each slide. Coverslips were applied and sealed as previously described. The slides were codenatured at 75°C for 1 minute and hybridized at 42°C for 1 hour. For the bcr/abl mixes, the slides were placed on a 37°C HYBrite, and the probe hybridization mixture was applied to each slide. Coverslips were applied and sealed as previously described. The specimen and probe were codenatured at 73°C for 5 minutes and hybridized at 37°C for 17 hours. Pretreated uncultured primary amniocytes were hybridized with the AneuVysion Assay. AneuVysion consists of two mixes that are hybridized on the same slide but under different coverslips. The first mix contains CEP Y SpectrumOrange, CEP X Spectrum-Green, and CEP 18 SpectrumAqua. The
Table 5. Comparison of VP 2000 and Manual Protocols for Cultured Lymphocyte Pretreatment VP 2000 Step 1 2 3 4 5 6 7 8 9
Manual
Description
Time (min)
Step
Description
Time (min)
37°C 2X SSC incubation 1% Formaldehyde/0.018 M MgCl2 incubation Continuous-flow distilled water rinse 37°C Protease II incubation Continuous-flow distilled water rinse 70% Ethanol dehydration 85% Ethanol dehydration 95% Ethanol dehydration 25°C dry
10 15 3 13 3 1 1 1 3
1 2 3 4 5 6 7 8 9
37°C 2X SSC incubation 1% Formaldehyde/0.018 M MgCl2 incubation 2X SSC 37°C Protease II incubation 2X SSC 70% Ethanol dehydration 85% Ethanol dehydration 100% Ethanol dehydration Ambient air-dry
10 15 5 13 5 1 1 1 5
SSC, standard saline citrate.
214 Molecular Diagnosis Vol. 5 No. 3 September 2000
second mix contains LSI 21 SpectrumOrange and LSI 13 SpectrumGreen. The slides were placed on a 37°C HYBrite, and 10 L each hybridization mixture was applied to each slide. The slides were codenatured at 73°C for 5 minutes and hybridized at 37°C for 6 hours. Pretreated uroepithelial cells were hybridized with UroVision Multicolor Probe, which contains CEP 3 SpectrumRed, CEP 7 SpectrumGreen, CEP 17 SpectrumAqua, and LSI 9p21 SpectrumGold™. The 12-circle slides were placed on a 39°C HYBrite, and 3 L hybridization mixture was applied to each circle that contained a specimen. Coverslips were applied and sealed as previously described. There were two to four circles with hybridization buffer underneath each coverslip. The specimen and probe were codenatured at 73°C for 2 minutes. Hybridization was at 39°C for 4 hours. Pretreated cultured lymphocytes were hybridized with TelVysion 17p SpectrumGreen, TelVysion 2p SpectrumGreen, TelVysion 3q SpectrumOrange,
TelVysion 21q SpectrumOrange, WCP 3 SpectrumGreen, and WCP 3 SpectrumOrange. The slides were placed on a 37°C HYBrite, and 10 L each hybridization mixture was applied to each slide. Coverslips were applied and sealed as previously described. The specimen and probes were codenatured at 70°C for 3 minutes. Telomere probes were hybridized at 37°C for 6 hours, and WCP probes were hybridized at 37°C for 16 hours. To remove nonspecifically bound probe, all specimen types were washed according to the manufacturer’s product insert. Specifically, after the slides were removed from the HYBrite, the coverslips were removed, and the slides were immediately placed in 73°C ± 1°C 0.4× SSC/0.3% Nonidet 40 in Coplin jars for 2 minutes. They were then placed in room temperature 2X SSC/0.1% Nonidet 40 for 1 minute, dried in the dark, and counterstained with DAPI II (Vysis, Inc, Downers Grove, IL). Visualization and scoring were performed us-
Fig. 2. Scoring criteria for FISH. (A) Example of a score of 2 on paraffin-embedded breast cancer section hybridized with FISH probes. For a passing score of 3, there should be clear, distinct signals in at least 80% of the cells. Nonspecific hybridization should be light enough and uniform enough to allow visualization of the signals. Here, less than 80% of the cells have distinct signals. Nonspecific hybridization is somewhat particulate and heavy enough to interfere with easy visualization of signals. (B) Example of a score of 4 on paraffin-embedded breast cancer section hybridized with FISH probes. For a score of 4, there should be clear, distinct signals in at least 90% of the cells. Nonspecific hybridization should be light enough and uniform enough to allow for easy visualization of the signals. Here, all cells in the same plane of focus have clear, distinct signals. There is light nonspecific hybridization in the background, but it does not interfere with visualization of the signals.
Automation of FISH Pretreatment
ing the manufacturer’s suggested filter sets appropriate for each test. Slide Evaluation Slides were rated on a scale of 1 to 5 for quality of hybridization, with 5 the highest possible score. A slide must have a score of 3 or greater for all evaluated parameters to be of acceptable quality. A score of 5 corresponds to exceptional quality, 4 corresponds to high quality, 3 corresponds to a slide that can be enumerated with reasonable ease, 2 corresponds to a slide in which signal is visible but not easily enumerated, and 1 corresponds to hybridization failure. Examples of a score of 4 and 2 are
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Jacobson et al. 215
shown in Fig. 2. Parameters evaluated were signal intensity, signal specificity, general background, cross-hybridization, and overall quality. Specimen loss was also evaluated for paraffin-embedded breast tissue sections. A score of 5 corresponded to no tissue loss; 4, less than 10% loss; 3, 11% to 25% loss; 2, 26% to 75% loss; and 1, greater than 75% loss. Greater than 10% tissue loss was seen on only a few specimens. Two raters independently evaluated each slide, and slides were blinded to type of pretreatment. The scores were analyzed using JMP (SAS Institute, Inc, Cary, NC) statistical software program and analyzed for significance using Student’s t-test. P less than .05 and R2 greater than 0.81 is considered statistically significant.
Table 6. Manual versus Automation Using the VP 2000: Specimen Pretreatment of Paraffin Section Slide Before Vysis FISH Manual Method
Incubation Time (min)
1. Reagent preparation and start-up Reagent prep 30 Load slides 1 Subtotal 2. Deparaffinization Hemo-De Hemo-De Hemo-De Ethanol Ethanol Subtotal 3. Pretreatment 0.2 N HCl Purified H2O Wash 2X SSC Pretreatment Purified H2O Wash 2X SSC Wash 2X SSC Subtotal 4. Enzyme digestion Protease Wash 2X SSC Wash 2X SSC Subtotal 5. Fixation 10% Formalin Wash 2X SSC Wash 2X SSC Subtotal 6. Dry slide Subtotal Total
31 10 10 10 5 5 40 20 3 3 30 1 5 5 67 10 5 5 20
Captive Time (min) 30 31
45
27
25
10 5 5 20 5 5 183
SSC, standard saline citrate.
25 1 0 154
VP 2000 Automation 1. Reagent preparation and start-up Reagent prep VP 2000 Processor start up Load slides Subtotal 2. Deparaffinization Hemo-De Hemo-De Hemo-De EtOH EtOH Subtotal 3. Pretreatment 0.2 N HCl Water rinse Pretreatment reagent Water rinse
Incubation Time (min)
Captive Time (min)
10 1 3 14
10 1 14
5 5 5 1 1 17
0
20 3 30 3
Subtotal 4. Enzyme digestion Protease I solution Water rinse
56
0
Subtotal 5. Fixation 10% Formalin Water rinse 70% ETOH 85% ETOH 100% ETOH Subtotal 6. Dry slide Subtotal
13
0
10 3 1 1 1 16 3 3 119
0 0 0 14
10 3
216 Molecular Diagnosis Vol. 5 No. 3 September 2000
Results Time Savings A comparison of the individual protocols for the VP 2000 and manual format is listed in Tables 1 through 5. As well as the time savings afforded by the walk-away feature of the automated protocol, the actual time to completion is significantly shortened in some protocols with automation. The optimal VP 2000 protocol was developed through empirical validation on different sample types. The protocol modifications in the VP 2000 were made for two primary reasons: total run time and reagent stability. In the automated protocol, many steps were shortened because an equivalent level of performance was achieved. This was because of the greater ratio of reagent to sample, resulting in a more effective dilution or treatment. Typically, a Coplin jar containing 50 mL reagent could process up to six slides, whereas the VP 2000 basins hold approximately 650 mL, for up to 50 slides. The second reason for the modifications is reagent stability. There is significant carry over of reagents with a fully loaded slide carrier. Although all reagents have been optimized with respect to concentration and pH, certain reagents, such as the 0.2 N HCl, pretreatment reagent, and proteases, are particularly sensitive to dilution effects and pH changes from the preceding reagent, which is typically 2X SSC. Prior rinsing of the sample slides in deionized water resulted in a significant improvement in reagent stability. In the case of amniocytes, manual 2X SSC pretreatment step no. 1 was kept at 37°C for 60 minutes because it follows the stated protocol in the package insert. However, manual
pretreatment at 73°C for 2 minutes works equivalently. A significant advantage of the VP 2000 Processor is the reduction in hands-on labor that it potentially provides to the laboratory. By reducing handson labor requirements in specimen pretreatment procedures, the technologist is freed to perform other tasks, providing greater efficiency and use of resources. This may allow resource-constrained laboratories a feasible solution for adopting FISH assays into their laboratories As described previously, certain tissue specimens must undergo a pretreatment process before performing the FISH assay. One particular specimen type that requires pretreatment is sectioned paraffin-embedded tissue. To prepare this type of specimen for the FISH assay, the specimen must undergo deparaffinization to dissolve the paraffin surrounding the tissue, followed by an enzymatic digestion to minimize proteins and connective tissue in and surrounding the cells. This procedure consists of 18 manual steps and takes approximately 183 minutes to complete. Manual processing using this procedure requires movement of the slides through a variety of reagents, incubating the slides in the reagents for specified periods of time, and then moving them to the next reagent. The incubation times associated with each step of the process range from 1 to 30 minutes, often leaving little free time for the technologist to perform other tasks within the laboratory while using this process. Use of the VP 2000 for the pretreatment process essentially eliminates all hands-on processing, thus freeing the technologist to perform other analyses or tasks. A comparative analysis of processing and incubation time and labor costs for the manual method
Table 7. Hands-on Time Savings Per Slide From VP 2000 Pretreatment VP 2000
Sample Type Uncultured amniocytes Bone marrow WCPs/TelVysion Uroepitheliel cells
Manual
Run Time (min)
Hands-on Time (min)
Total Hands-on Time/Slide (min)
Run Time (min)
Hands-on Time (min)
Total Hands-on Time/Slide (min)
Reduction in Processing Time/Slide (%)
24 24 47 32
30 30 30 30
0.6 0.6 0.6 0.6
96 + 30* 22 + 30* 56 + 30* 33 + 30*
146 72 106 83
2.9 1.4 2.1 1.7
79 57 71 65
Assay time is based on a batch size of 50 slides. For the VP 2000 protocol, hands-on time includes reagent preparation, loading slide rack, and selecting and starting a program. For the manual assay, hands-on time includes reagent preparation, incubation time, and slide handling during run. *Time required for slide handling.
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Jacobson et al. 217
Table 8. Results for PathVysion (HER-2) Mean Score Attribute
Range
VP 2000
Manual
VP 2000
Manual
Significance
3.88 3.91 4.11 4.20 4.78
3.27 3.39 3.83 4.19 4.37
3.00–4.25 3.25–4.25 3.75–4.25 3.50–4.35 4.00–5.00
1.75–4.00 2.00–4.13 2.75–4.50 3.50–4.50 1.50–5.00
.0000 .0000 .0000 NS .0000
Overall readability Probe intensity Probe specificity Background Tissue loss
All specimens were run pairwise. N ⳱ 16. NS, not significant.
versus the VP 2000 automated method for pretreatment procedure is listed in Table 6 . This analysis provides an estimated time comparison based on experience with both manual and automated methods as performed at Vysis, Inc. All time data points are recorded in minutes. For purposes of the analysis, the procedure was divided into six major sections: (1) reagent preparation and start-up, (2) deparaffinization, (3) pretreatment, (4) enzyme digestion, (5) fixation, and (6) drying. Within each section, the reagent used in the proFig. 3. Comparison of hybridization quality using manual and VP 2000 pretreatment protocols. (A) HER-2–amplified paraffin-embedded breast tumor section processed with manual pretreatment before hybridization. HER-2 probe is orange, and CEP 17 is green. Nuclear staining is blue (DAPI). Larger nuclei with multiple HER-2 signals are malignant, and smaller cells with one to two copies of each probe are normal. Some nuclei have been truncated by sectioning and thus have fewer than two copies of each probe. Signals can be enumerated, but not easily, as for the section on the right that was processed on the VP 2000. (B) HER-2–amplified paraffin-embedded breast tumor section from the same patient processed with VP 2000 pretreatment before hybridization. HER-2 probe is orange, and CEP 17 is green. Nuclear staining is blue (DAPI). Visualization of fluorophores is easier because more efficient digestion reduces autofluorescence of extracellular elements.
cedural step, incubation time (time that the slide remains in the reagent), and captive time (time that the technologist is practically required to perform the task or wait for the task to be completed) is described. Each procedure section is then subtotaled to provide information on time expended for that particular section of the process. The captive time for each step represents the true labor associated with the process. In cases in which the incubation time is short (ⱕ10 minutes), it is assumed that it would not be practical for the technologist to aban-
218 Molecular Diagnosis Vol. 5 No. 3 September 2000 Table 9. Results for AneuVysion Mean Scores Probe CEP 18, X, and Y LSI 13 and 21
Range
VP 2000
Manual
VP 2000
Manual
Significance
4.19 3.50
3.94 3.44
4.00–4.50 3.25–3.75
3.50–4.25 3.00–3.75
NS NS
All specimens were run pairwise. N ⳱ 8. NS, not significant.
don the process to perform other laboratory tasks; therefore, they are committed to the entire time span for that step. In some procedures, the steps of the manual method do not match the VP 2000 Processor method because the procedures are different. The total time for each method represents the time in minutes for the entire manual or VP 2000 process, incubation, and captive or labor time. The manual method for this procedure results in an incubation time of 183 minutes and a captive time of 154 minutes. The VP 2000 Processor automated procedure results in an incubation time of 119 minutes and captive time of only 14 minutes. A theoretical calculation of labor costs for this analysis based on an average hourly labor cost (including salary wage and benefits) of $40 results in a total labor cost of $102.66 for the manual procedure method and $9.33 for the VP 2000 method. Considering that the maximum number of slides that one would realistically be able to process per batch using the manual method is 10 slides, labor would cost $10.22 per slide in this scenario. In contrast, the VP 2000 Processor has the capacity to process 50 slides per batch, resulting in an extremely reduced labor cost of $0.19 per slide, which provides a significant cost savings to the laboratory. A summary of the time savings for other assays is listed in Table 7. Specifically, for a batch of 50 slides, the time savings ranged from 65% for uroepithelial
cells, 71% for lymphocytes, and 79% for amniocytes. Table 8 lists the results of pretreatment and hybridization on paraffin-embedded breast tissue sections using PathVysion HER-2. VP 2000 pretreatment resulted in statistically better results than the manual pretreatment protocol. This assay showed the greatest overall difference in performance between the VP 2000 and the manual format. Probe signal intensity and specificity were greater with the VP 2000 pretreatment than with the manual assay (Fig. 3). No tissue loss greater than 10% was seen with VP 2000 pretreatment. Conversely, a few samples (three of 16 samples) showed extensive tissue loss with the manual protocol. Mistakes during handling, such as placing a slide in the wrong Coplin jar or scraping a specimen, are essentially eliminated using the VP 2000 Processor. Replacement of 2X SSC solution rinses with continuousflow water rinses may also have influenced the outcome. The continuous-flow water rinse allows for more complete washing of the specimen and eliminates contamination of reagents with 2X SSC. Table 9 lists the results of pretreatment and hybridization on uncultured primary amniocytes with AneuVysion. In this case, the results of VP 2000 pretreatment and manual pretreatment were equivalent. This is consistent with the lower complexity of the pretreatment assay compared with PathVysion
Table 10. Results for Bone Marrows Mean Scores Probe CEP 8 CEP 12 CEP X/Y LSI bcr/abl
Range
VP 2000
Manual
VP 2000
Manual
Significance
3.81 3.37 3.81 2.88
3.44 2.81 3.69 3.13
3.00–4.50 2.75–4.50 3.50–4.25 2.50–3.25
3.00–4.00 2.50–3.25 3.25–4.50 2.75–3.50
.0340 NS NS NS
All specimens were run pairwise. N ⳱ 4 for each probe set. NS, not significant.
Automation of FISH Pretreatment
•
Jacobson et al. 219
Table 11. Results for PathVysion Bladder Probe SpectrumGold LSI 9p21 SpectrumAqua CEP 17 SpectrumGreen CEP 7 SpectrumRed CEP 3
VP 2000 Mean Scores
Manual Mean Scores
Significance
3.47 4.48 4.50 4.76
3.27 4.42 4.40 4.71
.0051 .4139 .3219 .5644
All specimens were run pairwise. N ⳱ 15.
(HER-2). Table 10 lists the results of pretreatment and hybridization on bone marrow cells. CEP 8 was improved by VP 2000 treatment, and there was no difference between manual and VP 2000 pretreatment for CEP 12, CEP X/Y, and LSI bcr/abl. Again, the smaller differences between VP 2000 and manual assays are consistent with the relatively low complexity of this pretreatment. Table 11 lists the results of pretreatment and hybridization on uroepithelial cells with UroVysion. There were no differences with CEP 3, CEP 7, and CEP 17, but signal quality for LSI 9p21 was slightly better with the VP 2000 pretreatment. Table 12 lists the results of pretreatment and hybridization on cultured lymphocyte metaphase spreads with telomere probes and Chromosome Paints. WCP 3 SpectrumOrange and SpectrumGreen performed the same regardless of assay. TelVysion probes, 2p SpectrumGreen, 17p SpectrumGreen, and 21q SpectrumOrange were statistically significantly improved by the VP 2000 assay. Average scores for 3q SpectrumOrange were greater with the VP 2000 assay, but the difference was not statistically significant.
Discussion A variety of cell pretreatment protocols have been known to improve FISH results for the detec-
tion of chromosome copy number aberration. Formalin-fixed, paraffin-embedded tissues are particularly refractory to FISH hybridization [4,5]. The number of individual manipulations required to manually process large numbers of slides containing this sample type is demanding and can result in uneven pretreatment of specimens in the hands of inexperienced users. Slight variations in individual reagent exposures and manual manipulation of slides can result in a large cumulative variation in pretreatment efficacy. We have shown that automation of all pretreatment steps greatly enhances the performance of FISH for PathVysion HER-2, as well as for a variety of FISH probes. This is particularly true on marginal samples, which were deliberately included in this study to reflect the wide variation in sample quality. Some of these variables can be controlled in the hands of experienced users, such as the preparation of metaphase slides or lymphocytes. When specimens are prepared under optimal conditions, FISH performance is optimal [6,7]. However, in the case of archived specimens, such as paraffin-embedded formalin-fixed tissue, optimization of pretreatment is one of the few options available. This study clearly shows that the greatest benefit of automated pretreatment is seen with these difficult samples. This outcome suggests that difficult samples are not refractory to standard
Table 12. Results for TelVysion and WCPs Mean Scores Probe WCP 3, SpectrumOrange WCP 3, SpectrumGreen 2p, SpectrumGreen telomere 3q, SpectrumOrange telomere 17p, SpectrumGreen telomere 21q, SpectrumOrange telomere
Range
VP 2000
Manual
VP 2000
Manual
Significance
3.50 3.33 3.81 3.25 3.62 3.38
3.50 3.33 1.69 2.25 1.38 2.19
3.50 3.25–3.50 3.75–4.00 2.75–3.75 3.50–3.75 3.00–3.75
3.25–4.00 3.25–3.50 1.25–2.00 1.75–2.50 1.00–2.25 1.50–2.50
NS NS .000 NS .0000 .0000
All specimens were run pairwise. For the WCP probes, n ⳱ 3. For the telomere probes, n ⳱ 4. NS, not significant.
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Molecular Diagnosis Vol. 5 No. 3 September 2000
pretreatment regimens but are exquisitely sensitive to overtreatment or undertreatment. This hypothesis could explain our observations on the variability in assay performance of the manual format on marginal samples. In replicate adjacent sections from the tissue block, the manual format shows much greater variations between runs than the VP 2000. The other major benefits for automation of pretreatment are the significant reductions in labor and time to result. These savings can also be realized in the development of custom protocols for special applications. The movement of FISH technology toward full automation is likely to achieve the same benefits observed with immunohistochemistry assays in which artistry is removed and improvements in quality, speed, and reproducibility are necessary for improved adoption of the technology in the clinical laboratory [8]. Additionally, the achievement of this level of quality for FISH can provide a greater level of sensitivity and specificity with less ambiguity than other competing technologies. Received February 24, 2000. Received in revised form March 14, 2000. Accepted May 22, 2000.
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