Implementation of Virtual Microscope Slides in the Annual Pathobiology of Cancer Workshop Laboratory

(1)

Implementation of Virtual Microscope Slides

in the Annual Pathobiology of Cancer

Workshop Laboratory

FRED R. DEE, MD, JOHN M. LEHMAN, P

H

D, DAN CONSOER, BA,

TIMOTHY LEAVEN, MA, AND MICHAEL B. COHEN, MD

Virtual slides are digital facsimiles of glass microscope slides

that, when viewed with a pan and zoom viewer, can emulate viewing a glass slide with a traditional microscope. Based on successful im-plementation of virtual slides in medical student histology and pa-thology courses at the University of Iowa, we developed a plan to evaluate the use of virtual slides in the American Association for Cancer Research’s annual Pathobiology of Cancer Workshop. In this Workshop, nonphysician predoctoral students and postdoctoral fel-lows working in cancer research explore the morphological, clinical, and molecular aspects of human cancer. Over the course of a week, students examine approximately 100 glass slides in microscope lab-oratories, facilitated by senior cancer investigators. The goal of the present study was to evaluate virtual slides as a teaching modality in

these laboratories, not as a replacement for traditional microscopy, but rather in terms of their utility in facilitating student learning as they examine glass slides with a traditional microscope. Evaluation by questionnaire indicated that virtual slides enhanced students’ ability to grasp morphological features better than the traditional photomi-crographs. The results of this implementation suggest that virtual slide technology may be successfully extended to other educational venues where traditional microscopy and photomicrographs are cur-rently used. HUMPATHOL34:430-436. © 2003 Elsevier Inc. All rights reserved.

Key words:virtual slides, virtual microscopy, education, histopa-thology, cancer.

Abbreviation:AACR, American Association for Cancer Research. Virtual slides are digital facsimiles of the visual

content of glass microscope slides, acquired at a high magnification. Using a web-based zoom viewer, virtual slides can be magnified from⫻0.625 to⫻40 and moved in ax–y axis on the computer screen, thus emulating the examination of a glass slide with a traditional mi-croscope.1-6

Virtual slides are acquired by assembling digital images of adjacent fields in histological sections or fluid smears into a giant image montage using a computer-controlled scanning stage. Although this technology has existed for more than a decade, this process (also called whole-slide digitization) has been commercially available for standard desktop computing only since 1998, when sufficient memory and processing speed became available. Because virtual slides can capture the important visual information of a glass slide, they can be used in all of the educational venues in which glass slides and digital photomicrographs are used, without losing the ability inherent in traditional glass slide mi-croscopy to explore the entire microscopic section, identify structures and lesions, and demonstrate their relationships and variations.

In 1997, realizing the important contributions that virtual slides might make to education in microscopic anatomy and pathology, we implemented a plan, which was subsequently supported by a National Library of Medicine Information Systems Grant, to digitize all of the slides in the histology and pathology courses at the University of Iowa. These virtual slides were then suc-cessfully incorporated into the histology and pathology courses in 2000 and 2001, where they are used to augment traditional glass slide microscopy.3,7,8 _Based on this positive experience, we decided to evaluate virtual slides as a teaching modality in the microscope laboratory at the American Association for Cancer Re-search’s (AACR) annual Pathobiology of Cancer Work-shop, in which 3 of the authors serve as course faculty. The goal was not to replace the use of glass slides and microscopes, but rather to augment the ability of course faculty to facilitate learning as students examine cancer histopathology slides with traditional micros-copy.

The AACR Pathobiology of Cancer Workshop is a 1-week course that had its inception in 1978. Supported by the National Cancer Institute (NCI) since 1982, the Workshop provides scientists working in cancer re-search with an intensive experience in the morpholog-ical, clinmorpholog-ical, and molecular aspects of human cancer. The course attendees are primarily nonphysician pre-doctoral students and postpre-doctoral fellows, and 5% are senior scientists.

The Workshop consists of 7 half-day gross and microscopic pathology laboratories, interspersed with lectures on current concepts of cancer, including pap-illoma viruses, transgenic mouse models, cancer pro-gression, metastasis, stem cell origins of cancer, molec-ular diagnosis, cellmolec-ular and molecmolec-ular therapy, and clinical trials. Six groups of 18 students each attend a laboratory session devoted to normal histology and in-From the Department of Pathology, Carver College of Medicine,

University of Iowa, Iowa City, IA, and the Department of Pathology, Brody School of Medicine, East Carolina University, Greenville, NC. Accepted for publication March 13, 2003.

Supported by a National Library of Medicine Information Sys-tems Grant (G08 LM06904-02) and a grant from the National Cancer Institute (5R25CA058981-10).

Address correspondence and reprint requests to Fred R. Dee, MD, 100 Medical Laboratories, Room 1198, University of Iowa, Iowa City, IA 52242.

(2)

troduction to cancer, and then rotate through six 4-hour laboratory sessions covering cancers of the ma-jor organ systems. These 6 laboratory sessions are orga-nized as (1) hematopoietic tissues and AIDS; (2) gas-trointestinal tract; (3) childhood tumors and brain; (4) breast, uterus, cervix, and ovary; (5) male genitourinary tract; and (6) lung and skin. Each laboratory is staffed by at least 2 faculty members.

In 2002, the Workshop faculty comprised 15 senior cancer investigators from 7 different US medical cen-ters. Most of the 15 have taught in the Workshop for more than 3 years, and 1 faculty member has partici-pated since the course’s inception in 1978, lending stability to the Workshop.

Learning aids in the laboratories include single-headed binocular microscopes (1 per student), micro-scopic glass slide sets (approximately 14 slides per lab-oratory session), one double-headed microscope, gross specimens, overhead transparencies, 2 ⫻ 2 carousel slides, Microsoft PowerPoint (Microsoft Inc, Redmond, WA) presentations, and, more recently, virtual slides, which use the same computer projection equipment as PowerPoint. A typical laboratory is illustrated in Figure 1.

In the laboratories, using a traditional microscope and glass slides, students identify the characteristic find-ings of cancer including dysplasia, in situ carcinoma, invasion, metastasis, abnormal mitotic figures, hyper-chromatism, anaplasia, and differentiation, while com-paring and contrasting normal, hyperplastic and neo-plastic tissues. Students also identify the characteristics of individual cancer types for each organ system. Study-ing the histology and gross pathology of specimens concurrent with information related to the pathogene-sis and pathobiology and clinical features helps the students develop a global concept of cancer. Use of the

traditional microscope is an integral part of the course. This is not only because many of the trainees will be evaluating histologic slides in their own research labo-ratories, but also because the course faculty believe that the active learning associated with examining real glass slides is critical to developing an understanding of the pathobiology of cancer.

The typical laboratory is organized as follows. Fac-ulty members give a brief introduction to the concepts and content of the laboratory session, then introduce the clinical and gross features of the cancer, followed by the microscopic characteristics that the students are expected to find on their own glass slide. The instruc-tors then circulate to assist students as they examine the slides and to discuss concepts of cancer exemplified by the laboratory materials.

Many students in the Workshop have had a mini-mal amount of microscopic morphology. Thus the fac-ulty’s ability to demonstrate important features on the projector screen at the front of the laboratory is critical to the learning process. Before 2001, teaching aids for microscopy morphology consisted of 2⫻ 2 slides pro-jected by a carousel projector or PowerPoint computer projector. Videomicroscopy was occasionally used, be-cause it allowed the faculty to project the actual side the students were examining; however, this modality proved too cumbersome for routine use. The double-headed microscope is used sparingly for individual stu-dents when a specific issue requires direct interaction between a student and a laboratory director.

To test the feasibility of using virtual slides as a teaching aid in the Workshop laboratory, we imple-mented virtual slides and evaluated them in 1 labora-tory unit (male genitourinary tract) in the July 2001 laboratory. Based on the positive 2001 formative evalu-ation from this laboratory (described later in this arti-cle), we created a website for the course, and fully implemented virtual slides in all of the Workshop lab-oratories in July 2002.

MATERIALS AND METHODS

The method outlined here describes the technology used in 2001-2002 to develop virtual slides for the AACR course. It is similar to that used for other Iowa virtual slide educational programs (see http://www.path.uiowa.edu/ virtualslidebox/).3,7,8_{We anticipate that the technology used} for future iterations of the AACR course will change as virtual slide technology evolves.

Acquisition

Visual slides were created with a system using Virtual Slice (MicroBrightField, Williston, VT) and ScanScope (Ape-rio Technologies, Vista, CA) technologies. The system in-cluded a panning motor, autofocus high-resolution micro-scope lens, optical capture device, computer, and software that automatically coordinates the activities of the motor and video capture. The equivalent of⬃1200 contiguous⫻40 mi-croscope fields were automatically digitized, merged into 1 seamless image montage, and outputted as a single gigabyte-sized uncompressed .tif file of approximately 20,000⫻20,000 FIGURE 1. A Keystone laboratory in progress. Dual projectors

in this laboratory allowed projection of virtual slides and Pow-erPoint slides on the screen at the right, and gross images, diagrams or supplemental photomicrographs on the screen at the left. Students are seated at tables with microscopes and sets of glass slides. (This figure has been electronically en-hanced).

VIRTUAL MICROSCOPE SLIDES (Dee et al)

(3)

(4)

pixels. Each file captured approximately 0.9 cm2_{of the} sur-face of the glass slide. Actual digitization time ranged from about 15 to 90 minutes, depending on the equipment and configuration used. Although virtual slides prepared for the 2002 Workshop were limited in size and speed of acquisition by the technology in existence at that time, it is now possible to create files larger than 100,000⫻ 100,000 pixels. In the future, emerging acquisition technology that uses a multisen-sor design and miniaturized microscope arrays, may markedly decrease acquisition time, to less than 1 minute.6

Conversion

The single uncompressed image file was cropped and enhanced for brightness, contrast, and sharpness in Adobe Photoshop (Adobe Systems, San Jose, CA). This large file was then converted into a single multiresolution pyramidal file called flashpix. This .jpeg compression file contains multiple levels of resolution equivalent to⫻40, 20, 10, 5, 2.5, 1.25, and 0.625 magnification. Roxio/MGI software (Roxio Inc, Santa Clara, CA) was used for the conversion to flashpix.

Serving and Viewing

The compressed flashpix virtual slide files of approxi-mately 100 mB each were loaded onto a Dell PowerEdge 4400 computer (Dell, Round Rock, TX) running on a Linux op-erating system and Apache web server (Apache Digital, Du-rango, CO). Identifying information for each slide (slide number, diagnosis, and laboratory unit) was added to a MySQL database (MySQL AB, Uppsala, Sweden). Customized Perl CGI scripts were used to generate a table of contents for the virtual slides in each unit from the MySQL database and to deliver the images to the computer screen in html pages. Virtual slides were served with the MicroBrightField Virtual Slice system, which has as components MGI Zoom server software (Roxio) and a Java applet viewer imbedded in an web browser window (Fig 2). A zoom function in the viewer changes magnification by 2-fold with each click of the mouse by jumping from 1 resolution level to the next in the flashpix file. A click-and-drag function permits scanning of each mag-nification in anx–yaxis by streaming from the server to the viewer only the portion of the slide being viewed. Customized Perl CGI scripting is also used to direct a MicroBrightField java applet annotator to add arrow coordinates and text to the MySQL database, and subsequently to the slide when an html call is made by clicking on an annotation button (Fig 3A). A Perl CGI scripted side-by-side viewing function (Fig 3B) will be added to the Workshop laboratory site in 2003. This will allow comparison of cancer slides with normal, hyperplastic, and other cancer types.

Once the slides and database information for the Work-shop laboratory were prepared and added to the server, a Workshop laboratory website was generated from the data-base and made available to faculty before the course started. Because the Workshop site in Keystone does not have web access in each laboratory, Dell Inspiron 8500 laptop comput-ers, running Microsoft XP (instead of Linux), were loaded with the flashpix virtual slide files and software (Apache server, the Perl scripted MySQL database, and MicroBright-Field Virtual Slice server and viewer system), allowing them to run as local-host web servers in the Workshop laboratories.

In July 2001, one Workshop laboratory (male genitouri-nary tract) was equipped with a laptop computer containing virtual slides and a computer projector. The 5 other labora-tories continued using traditional photomicrographs pro-jected with a 2⫻2 carousel or Microsoft PowerPoint with a laptop and computer projector. Faculty in the male genito-urinary tract laboratory used the virtual slides to demonstrate structures and lesions that students were expected to observe under a microscope. At the end of the laboratory sessions, the students were asked to complete a formative evaluation form that asked them to compare the teaching value of virtual slides in this laboratory versus the projected photomicro-graphs in the other laboratories.

In July 2002, all of the 6 laboratories were equipped with virtual slides.

RESULTS

The evaluation data from July 2001, shown in Ta-ble 1, indicate that, compared to photomicrographs, virtual slides (1) were of higher image quality and resolution, (2) allowed the faculty to better point out cells and lesions that students were expected to see on the glass slide, and (3) enhanced the students’ ability to learn from glass slides at the microscope.

Student comments in support of virtual slides in-cluded “It really helped to have instructors find struc-tures,” “I was better able to identify thespecificfeatures on my slide,” “This definitely helped with identifying lesions better than normal projector slides,” and they “helped (me) to know that I was looking at the correct structures.” Some criticisms of the virtual slides are that they “can make you lazy” and that some students “didn’t look in detail at their own slides.”

In July 2002, based on the positive formative eval-uations obtained in July 2001, all 6 Workshop labora-tories were equipped for virtual slide projection. Al-though it was not possible to make comparisons again, positive comments about the use of virtual slides mir-rored those of 2001. Similar to previous years, the Workshop laboratories were evaluated by the students as being very successful. For example, 91% of students strongly agreed that their knowledge of the histopathol-ogy of cancer increased. The mean ratings for both the lectures and the laboratories in 2002 are shown in Table 2. Note that the highest average student ratings in 2002 were for the laboratory subject matter (4.85) and quality of the slides (4.64). Additionally, the entire faculty agreed that the virtual microscope was a useful and successful addition to the laboratories.

DISCUSSION

The longevity of the AACR’s annual Pathobiology of Cancer Workshop is but one indicator of its success. Since its inception in 1978, the Workshop has been

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™

FIGURE 2. a. (top) Low power (5x) view of a virtual slide. 2b. (bottom) High power (40x) view of a virtual slide. Magnifications of 0.625x, 1.25x, 2.5x, 10x, and 20x are also possible. Each magnification can be moved in the x-y axis by clicking and dragging the image.

(5)

(6)

based on teaching in small groups with a student to faculty ratio of about 8:1, which necessarily involves active participation by all. The advent of new technol-ogies has brought real opportunities to enhance the students’ learning experiences. Even after only one year of implementation, the virtual slides appear to have had a significant impact on the educational envi-ronment over the previously used approach.

The microscope teaching strategies used in the Workshop laboratories, now augmented with virtual slides, are similar to those used in many medical stu-dent pathology and histology course laboratories. Thus, we believe that virtual slide technology could be imple-mented in these traditional laboratory venues to re-place both 2⫻2 photomicrographs and videomicros-copy, 2 modalities commonly used to augment student learning in microscope laboratories and small groups. The advantages of virtual slides are summarized in the following paragraphs.

As opposed to the selective field of view of pho-tomicrographs, an entire facsimile of a slide can be projected, magnified, and moved on thex–yaxis. This allows students to orient themselves on their slides with respect to the slide being projected. Note that in Figure 2, the screen retains a whole mount of the glass slide and a thumbnail, with a navigation box indicating the area of the slide being viewed. This greatly assists the faculty in navigating the slide, and indicates to students where they should be looking on their own slides. To paraphrase one Workshop student, “virtual slides are the digital equivalent of a multiheaded microscope for the entire class.” Moreover, each student simulta-neously has his or her own microscope. Compared with the virtual slides system, in videomicroscopy the video camera has a very small low-power field of view (usually giving no lower than the equivalent of⫻5) and there is no thumbnail with a navigation box.

An additional useful feature of virtual slides is the ability to preselect fields of view and add arrows. This feature (Fig 3A) allows the faculty to locate difficult-to-find structures (eg, Reed-Sternberg cells, tripolar mi-totic figures) before a laboratory session, and then easily retrieve them in the laboratory session by clicking on an annotation button. Unlike videomicroscopy, vir-tual slides can be viewed side-by-side (Fig 3B), allowing simultaneous comparison of different pathologic

le-sions and normal with abnormal tissue. Finally, the image quality of the digital virtual slides is far superior to that of videomicroscope images.

Virtual slides have applications in settings other than the traditional microscope laboratory. At the Uni-versity of Iowa, both histology laboratories and pathol-ogy case analysis exercises are now taught in combined computer and microscope laboratories and in small group rooms equipped with both virtual slides and videomicroscopy equipment.3,7,8 _{In these settings,} stu-dents can view the glass teaching slides with the tradi-tional microscope, with videomicroscopy, or as virtual slides on the computer. From these options, students markedly prefer using virtual slides to prepare for small group sessions, and pathology faculty and students alike prefer using virtual slides for small group discussions. Major advantages of virtual slides from the student’s perspective include efficiency and accessibility. In terms of efficiency, all of the slides are accessible, in focus, and with proper lighting and condenser adjustment, at the click of a mouse. The slides can also be integrated into html pages with patient information, gross images, and radiological images, and linked to other web-based resources. All of these features markedly facilitate stu-dent presentation of microscopic and other findings in small groups. Accessibility of web-based virtual slides allows students to prepare outside of scheduled labora-tory time in computer laboratories, in student commu-nities equipped with computers, and at home with a high-speed Internet connection (cable or DSL). Stu-dents can also study together at the computer screen in groups of their own choosing anywhere and anytime. Faculty also benefit from virtual slide technology in that they can prepare for teaching over the web; project virtual slides in auditoriums, laboratories, and small groups; and better interact with students as they evalu-ate virtual slides on the computer or projector screen. Faculty can also easily download screen shots of virtual slides or link to the source code of individual virtual slides for use in other computer-based educational pro-grams.

Outside of laboratory and small group teaching, emerging virtual slide technology has significant poten-tial for innovation wherever traditional microscopy and photomicrographs are used. These venues include web-based publishing of atlases, textbooks, and articles;

con-4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™

FIGURE 3. a. (top) Annotated virtual slide at 40x. The arrows point at Reed Sternberg cells. 3b. (bottom) Side-by-side view of papillary carcinoma and normal thyroid. The bottom frame appears when theNormal histology Thyroidlink is activated.

TABLE 1. Student Comparison of Photomicrographs and Virtual Slides in 2001 (n⫽84)

Compared to photomicrographs, the virtual slides. . .

Strongly

Agree Agree

About

the Same Disagree

Strongly Disagree

Were of high image quality and resolution 59 21 4 0 0

Enhanced the instructor’s ability to point out cells and lesions that you were

expected to see on the glass slide 74 9 1 0 0

Enhanced your overall ability to learn

from the glass slides at the microscope 63 14 6 1 0

(7)

tinuing medical education, computer-assisted case sim-ulations and adaptive learning exercises; proficiency testing and certifying examinations; and telepathol-ogy.6 _{Many of these venues for virtual slides were} re-cently presented, displayed, and discussed at the Inau-gural Symposium on Virtual Slides at the Medical University of South Carolina, Charleston, SC, November 2002 (more information available at http://histology. musc.edu/vslide).

A National Library of Medicine Information Sys-tems Grant has allowed us to develop a public access database of histology and pathology virtual slides, the “Virtual Slidebox of Histopathology” (see http://www. path.uiowa.edu/virtualslidebox/ for more informa-tion). The Virtual Slidebox currently contains more than 600 virtual slides, including many from the AACR Workshop as well as many contributed by educators at other institutions. Initially, the content of the Virtual Slidebox relied on a core list of morphological entities of disease published in HUMANPATHOLOGYin 1998.9

Public web access to slides in the database is avail-able to course directors via an html link, allowing them to display virtual slides in their own html web pages. Also, course directors can request that virtual slides be mailed as tiff or flashpix files via removable media to serve from their own server. Because most of the orig-inally acquired files are in a tiff file format, they can be edited and adapted by others for use in a wide variety of multiresolution pyramidal file server and viewer config-urations.

In summary, the evaluation of virtual slides in the Pathobiology of Cancer Workshop laboratories indi-cated that students were better able to grasp the mor-phological features of cancer in their glass slide sets when instructors used virtual slides rather than tradi-tional photomicrographs to guide them. Although the initial cost of equipment and software for creating and delivering virtual slides is quite high, and industry stan-dards have not yet crystallized, we believe that this new technology has the potential to revolutionize the way we teach and learn from microscopic images. Our hope

is that virtual slide technology will be promoted and implemented in all of the various venues for micros-copy, thus stimulating healthy competition among vir-tual slide developers as they continue to improve the quality of their virtual slide scanners, servers, and view-ers. The end result should be that virtual slide technol-ogy rapidly becomes effective, efficient, and affordable for all educators involved in teaching microscopic anat-omy and pathology.

Acknowledgement. The authors gratefully acknowledge the support of the following Workshop faculty members who in 2002 helped create the AACR virtual slide laboratories and implemented them in their laboratory sessions:

Jeffrey M. Arbeit, MD University of California, San Francisco Stephen Baird, MD VA Medical Center, San Diego Michael B. Cohen, MD University of Iowa Health Care Carlyne Cool, MD University of Colorado Health

Sciences Center

Barry R. De Young, MD University of Iowa Health Care Fred R. Dee, MD University of Iowa Health Care Bette K. DeMasters, MD University of Colorado Health

Sciences Center

Roy A. Jensen, MD Vanderbilt University Medical Center John M. Lehman, PhD Brody School of Medicine East

Carolina University Robert L. Low, MD, PhD University of Colorado Health

Sciences Center Stewart Sell, MD Albany Medical College

David S. Strayer, PhD, MD Thomas Jefferson University Medical Center

Ann D. Thor, MD University of Oklahoma Health Sciences Center

Frederic M. Waldman, PhD, MD

University of California, San Francisco Cancer Center

Jianzhou Wang, MD, PhD University of Oklahoma Health Sciences Center

The authors also thank Nadine Lomakin, Program Adminis-trator, American Association for Cancer Research, for collect-ing evaluation data and coordinatcollect-ing the preparation of Workshop laboratories for virtual slide projection.

REFERENCES

1. Silage DA, Gil J: Digital image tiles: A method for the pro-cessing of large sections. J Microsc 138:221-227, 1985

2. Westerkamp D, Gahm T: Non-distorted assemblage of the digital images of adjacent fields in histological sections. Anal Cell Pathol 5:235-247, 1993

3. Harris T, Leaven T, Heidger P: Comparison of a virtual mi-croscope versus a regular mimi-croscope laboratory for teaching histol-ogy. Anat Rec 265:10-14, 2001

4. Steinberg DM, Ali SZ: Application of virtual microscopy in clinical cytopathology. Diagn Cytopathol 25:389-96, 2001

5. Leong FJ, McGee JO: Automated complete slide digitization: A medium for simultaneous viewing by multiple pathologists. J Pathol 195:508-514, 2001

6. Weinstein RS, Descour MR, Liang C: Telepathology overview: From concept to implementation. HUMPATHOL32:1283-1299, 2001

7. Dick (Dee) FR: Web-based virtual microscope laboratories. Pathol Educ 25:58-62, 2001

8. Heidger P, Dee FR, Consoer D: An integrated approach to teaching and testing in histology with real and virtual imaging. Anat Rec 269:107-112, 2002

9. Dillman D, Torner R, Finken L: Core morphologic concepts of disease for second-year medical students. HUMPATHOL 29:1017-1020, 1998

TABLE 2. Student Evaluation of Lectures and

Laboratories in 2002 (n⫽98)

Mean SD The lecture subject matter was interesting and

valuable. 4.55 .70

The lecture subject matter was largely new to

me. 3.92 1.10

The lectures put a lot of emphasis on general principles that are broadly applicable to

cancer. 4.46 .80

The laboratory subject matter was interesting

and valuable. 4.85 .39

The laboratory subject matter was largely new to

me. 4.54 .78

The quality of the slides was excellent. 4.64 .62 NOTE. Student responses rated on a scale of 1 (strongly dis-agree), 2 (somewhat disdis-agree), 3 (neutral), 4 (somewhat dis-agree), and 5 (strongly agree).