Michael L. Astion, 3 Joseph Rank,1 Mark H. Wener, Paul Torvik, Joe B. Schneider,1 and Lawrence M. Kilhingsworth 2
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1 CLIN. CHEM. 41/9, (1995) #{149} Oak Ridge Conference Electrophoresis-Tutor: An Image-Based Personal Corn puter Program That Teaches Clinical Interpretation of Protein Electrophoresis Patterns of Serum, Urine, and Cerebrospinal Fluid Michael L. Astion, 3 Joseph Rank,1 Mark H. Wener, Paul Torvik, Joe B. Schneider,1 and Lawrence M. Kilhingsworth 2 High-resolution protein electrophoresis of serum, urine, and cerebrospinal fluid (CSF) can aid in the diagnosis of multiple myeloma, amyloidosis, macroglobulinemia, multiple sclerosis, and other diseases. Electrophoresis-Tutor is a personal computer program based on -150 digital images that teaches the clinical interpretation of agarose gel electrophoretic patterns. The program is divided into the following sections: introduction, CSF, serum, urine, review of disease states, program navigator, and final exam. The CSF section describes normal and abnormal CSF findings with emphasis on oligoclonal banding, as seen in the CSF of patients with multiple sclerosis. The serum section emphasizes monoclonal gammopathy patterns but also has detailed descriptions of inflammation, liver disease, protein-losing disorders, genetic deficiencies, and other patterns. Monoclonal gammopathy is described in the context of specific associated clinical conditions (e.g., myeloma, amyloidosis). For each monoclonal gammopathy example, results of standard electrophoresis, densitometry, and immunofixation are presented. The review of disease states uses animation to illustrate the development and remission of a variety of pathological patterns. The program navigator allows the user to jump quickly to any place in the program. The optional exam contains 20 questions, and detailed feedback is given after each question. Electrophoresis-Tutor can be used as a stand-alone teaching tool, a companion to traditional instruction, or a reference source. High-resolution protein electrophoresis of serum and urine is an aid in the diagnosis of a variety of disease states, most notably multiple myeloma and related plasma cell disorders (1). Protein electrophoresis of cerebrospinal fluid (CSF) can help in the diagnosis of multiple sclerosis (2). Urine electrophoresis is useful in characterizing the type of proteinuria and in diagnosing and monitoring monoclonal gammopathies (2, 3). The standard methods for teaching the interpretation of protein patterns are supervised instruction at the laboratory bench and textbook instruction based on photographs. The major drawbacks to supervised in- Department of Laboratory Medicine, 1 University of Washington, Box , Seattle, WA 98195, and 2 Sacred Heart Medical Center, Spokane, WA. 3Author for correspondence. Fax Received April 20, 1995; accepted June 13, struction are teacher-to-teacher variability and lack of available time and expertise to teach the full range of clinically significant findings. Textbooks are very useful references (4-6). However, it is unrealistic to expect the majority of medical technology students, medical students, and other trainees to read an entire textbook about clinical electrophoresis. In addition, it is sometimes difficult to produce photographs that accurately represent subtle aspects of a stained gel. High-resolution images that faithfully represent protein staining patterns can now be captured inexpensively by digitizing an image of the gel through use of a flat-bed scanner interfaced to a personal computer (PC). Most PCs are capable of displaying these digital images. Here we describe a computer program, Electrophoresis-TutorTM (Beckman Instruments, Brea, CA), that uses these images to teach the clinical interpretation of protein staining patterns from serum, urine, and CSF. The tutorial, based on -150 highquality images of high-resolution electrophoresis patterns, is the latest in our series of computer programs that teach the interpretation of image-based clinical laboratory tests (7-10). Materials and Methods Electrophoresis-Tutor was designed, written, and tested by an experienced team of technologists, MDs, PhDs, and computer programers. The digital images in the program were derived from patients specimens submitted for serum, urine, or CSF electrophoresis to either the clinical chemistry laboratory at Sacred Heart Medical Center (Spokane, WA) or the clinical immunology laboratory at the University of Washington Medical Center (Seattle, WA). The specimens were electrophoresed with the Paragon electrophoresis system [serum protein electrophoresis kit or immunofixation electrophoresis kit; Beckman Instruments], according to the manufacturer s instructions, or with an in-house system in use at Sacred Heart Medical Center and the University of Washington. For the in-house method, electrophoresis of proteins in serum, CSF, and urine was carried out in a 1.0% medium electroendosmosis agarose gel (FMC Seakem Agarose MEEO on FMC Gelbond Film; FMC, Rockland, ME), 0.75 mm thick. The electrophoretic 1328 CLINICAL CHEMISTRY, Vol. 41, No. 9, 1995
2 buffer was a ph 8.6 barbital buffer that contained calcium lactate. We applied 4-pL specimens to the gel, using a plastic application template. Electrophoresis took place at 125 ma for -45 mm in a chamber that was water-cooled to a constant temperature of 4 #{176}C. The separated proteins were fixed with a solution of methanol and acetic acid, stained with amido black (serum), and then counterstained with Coomassie Blue (CSF and urine). The staining patterns present in the dried gels were digitized (TIFF format) by using a digital imaging system consisting of a 300 dpi, black and white, flat-bed scanner (Apple Scanner; Apple Computer, Cupertino, CA) interfaced to an Apple Quadra 900 microcomputer. The system was driven by Ofoto scanning software (Light Source Computer Images, Larkspur, CA). The images were transferred in TIFF format to an IBM-compatible PC (Gateway, Sioux Falls, SD). When appropriate, the images were edited by using Adobe Photoshop for Windows (Adobe Systems, Mountain View, CA). An early version of Electrophoresis-Tutor was reviewed at the University of Washington, the Sacred Heart Medical Center, and Beckman Instruments by 15 medical technologists as well as by a group of medical technology students, pathology residents, and faculty. These users provided detailed feedback that helped us extensively revise the tutorial. Program Description Electrophoresis-Tutor is written in Microsoft Visual Basic for Windows (Microsoft, Redmond, WA), a visual programing language (11). The tutorial requires an or Pentium PC (IBM compatible) with 4 Mbytes of RAM, 25 Mbytes of hard-disk storage, and a super video graphics adapter (SVGA) capable of showing 256 colors at a display resolution of 800 x 600 pixels. The program is intended for medical technologists, medical technology students, MDs, PhDs, and other healthcare workers or trainees. Designed for users with minimal computer experience, it is operated completely by pointing with the mouse and pressing the left mouse button. A schematic of the program is presented in Fig. 1. The program is divided into the following sections: introduction, cerebrospinal fluid, serum (with or without urine), urine, review of disease states, program navigator, and the final examination. The introduction describes the techniques of high-resolution protein electrophoresis in agarose, immunofixation, and densitometry and the general clinical use of these techniques. In addition, this section teaches some of the rudimentary knowledge needed to interpret clinical electrophoresis patterns. The introduction uses various teaching techniques, including animation to illustrate migration of proteins in an electric field, and graphic overlays that unambiguously highlight areas of interest in a gel. The CSF patterns section consists of a brief discussion of normal CSF findings and a longer section on abnormal CSF patterns. The abnormal CSF patterns focus on oligoclonal banding patterns, such as may be seen in multiple sclerosis-the primary use of CSF protein electrophoresis being as an aid in the diagnosis Fig. 1. Schematic diagram of the Electrophoresis-Tutor computer program. See text for details. CLINICAL CHEMISTRY, Vol. 41, No. 9,
3 Agaros. gel, serum Agerose gel, urine Immunoflxatlon, s.rum Immunofixatlon. urine D.nsltomstry. serum Example 11 1_Donsitometry, urine Quiz case summary Fig. 2. Schematic diagram of the multiple myeloma section of the program. An overview coversbasic facts about the disease.the 11 cases presented show the variety of electrophoresis findings that can be associated with this malignant condition. of multiple sclerosis. The tutorial points out that a finding of oligoclonal bands in CSF is -90% sensitive and 90% specific for this disease, and that infections and other diseases of the central nervous system are also associated with oligoclonal banding in CSF. The serum patterns section, by far the largest section in the tutorial, is divided into the following categories: monoclonal gammopathy, inflammation, liver disease, protein-losing disorders, genetic deficiencies, and other patterns. Monoclonal gammopathy is discussed in greatest detail, because the most significant use of serum protein electrophoresis is screening for monoclonal gammopathy in suspected cases of multiple myeloma and other plasma cell disorders. Monoclonal gammopathy is further classified by clinical state; this classification consists of multiple myeloma, primary Fig. 3. Two of the viewing options available for example 1 in the multiple myeloma section: (A) immunofixation electrophoresis for myeloma patient who has a large IgG-lambda monoclonal gammopathy; (B) densitometry results in register with the gels of the normal control (black) and the myeloma patient (mci) CLINICAL CHEMISTRY, Vol. 41, No. 9, 1995
4 Fig. 4. First (A) and last (B) screen from an animation sequence in the Review of Disease States section that shows transformation from a normal pattern to a liver disease pattern. amyloidosis, Waldenstrom macroglobulinemia, monoclonal gammopathy of uncertain significance, and others (e.g., solitary plasmacytoma, lymphoma, peripheral neuropathy). This classification is not meant to imply that serum protein electrophoresis can, by itself, differentiate these conditions; rather, the program emphasizes that a particular diagnosis is based on many clinical findings, including the electrophoresis results. In the monoclonal gammopathy section, the strategy for presenting each clinical condition is similar. This strategy is shown schematically in Fig. 2. The user is first given an overview of the characteristic features of the condition, including a description of the diagnostic and prognostic role of serum and (or) urine electrophoresis. After the overview, the user may view a variety of cases of electrophoresis results observed in that condition. The multiple myeloma section contains 11 such cases. Some of the viewing options from example 1 in the multiple myeloma section are shown in Fig. 3. The patient in this example has a large (>20 g/l) IgG lambda monoclonal gammopathy in the gamma region with a surrounding hypogammaglobulinemia. The example can be viewed in the following ways: (a) two adjacent lanes from an agarose gel, one lane derived from a disease-free control and one lane derived from the patient with myeloma; (b) the immunofixation results derived from the myeloma patient (Fig. 3A); (c) the same as case a, but also showing densitometry results in register with the gel results, the patient s results being shown in red and the control s in black (Fig. 3B). Each of the images is overlaid with descripfive text pointing out the key features of the image. In this example, the patient has neither a monoclonal component in the urine nor proteinuria, so the urine result is not shown. In other cases, the examination of urine is essential and the user has, in addition to all the serum displays discussed above, the option to view the results of urine electrophoresis, urine immunofixation, and urine densitometry, all with appropriate CLINICAL CHEMISTRY, Vol. 41, No. 9,
5 controls. Complex cases are always accompanied by a case summary. The serum section also contains subsections covering inflammation, liver disease, and protein-losing disorders. As in the monoclonal gammopathy subsection, these topics consist of an overview and at least two examples. The last serum subsection consists of other patterns : normal plasma, variations in complement, hemolysis (both intravascular and in vitro), and genetic variants such as a1-antitrypsin deficiency. The urine section shows examples of glomerular proteinuria, tubular proteinuria, and mixed glomerular and tubular proteinuria. The review of disease states uses animation to illustrate the development and remission of various pathological patterns, as illustrated in Fig. 4. Fig. 4A shows the first screen encountered if the user chooses to review the liver disease pattern. This screen consists of a normal serum pattern and two buttons, one labeled show liver disease, and the other labeled show normal. After the show liver disease button is activated, the pattern gradually changes from the normal pattern to a typical example of the pattern of chronic liver disease (Fig. 4B). The pattern of chronic liver disease is characterized by anodal slurring of the albumin band secondary to bilirubin binding, marked polyclonal hypergammaglobulinemia, and a prominent betagamma bridge consisting mostly of IgA. The pattern gradually returns to normal (Fig. 4A) after the user activates the show normal button. The other disease states that can be reviewed by similar methods are monoclonal gammopathy-myeloma type (i.e., large monoclonal gammopathy with surrounding hypogammaglobulinemia), acute inflammation, chronic inflammation, and CSF oligoclonal banding. The program navigator is an interactive map that allows the user to have one-touch access to any place in the program without having to go through any layers of the program. This makes Electrophoresis-Thtor an easy to use reference source because users can rapidly display any image in the program. There is a program navigator button at every major menu in the program. The final examination consists of 20 image-based, multiple-choice questions that cover topics contained in Electrophoresis-Tutor. The questions are presented in random order. Correct answers with explanations are provided for each question. There are two question formats. The first consists of a question with five possible answers, only one of which is correct. The second format consists of two images and a statement; the student is asked whether the statement applies to image A, image B, both images, or neither image. Discussion In our clinical laboratories, we use Electrophoresis- Tutor as a supplement to supervised instruction. The trainees-whether doctors, technologists, or students-start the electrophoresis rotation by going through Electrophoresis-Tutor. This allows the trainees to come to the teaching bench prepared, so that instructors can proceed directly to in-depth coverage of interesting cases rather than having to teach very basic material. Both teachers and students gain because the students move substantially beyond the basics, and the teachers do not have to perform some of the more monotonous aspects of teaching. Another important use of Electrophoresis-Tutor is as a reference source for both instructors and students. We often clariqy cases at the bench by comparing them with cases in the program. The tutorials program navigator feature is particularly useful in this context. The approach to teaching embodied in the tutorial could be useful for teaching the interpretation of any electrophoresis-based laboratory test, e.g., hemoglobin electrophoresis, capillary zone electrophoresis, electrophoresis of nucleic acids, and others. Our plans include developing tutorials to cover some of these topics, updating Electrophoresis-Thtor, and studying the educational effectiveness of the tutorials. We thank Mary Tyllia and the staff of the immunology laboratory of Sacred Heart Medical Center, and the staffs of the computer section and clinical immunology laboratory of the University of Washington for help with all phases of this project. In addition, we thank Carl Joliff of Physicians Laboratory Services (Lincoln, NE) and Lydia Dodson-Lehrer of Beckman Instruments for help with the final revision of the tutorial. References 1. Gandara DR, Mackenzie MR. Differential diagnosis of monoclonal gammopathy. Med Clin North Ani 1988;72: Kilhingsworth LM. Clinical applications of protein determinations in biological fluids other than blood. Clin Chem 1982;28: Waller KV, Ward K, Mahan JD, Wismatt DK. Current concepts in proteinuria [Review]. Clin Chem 1989;35: Keren DF. High-resolution electrophoresis and immunofixation. Boston: Butterworths, 1987:238pp. 5. Epstein E, Karcher RE. Electrophoresis. In: Burtis CA, Ashwood ER, eds. Tietz textbook of clinical chemistry, 2nd ed., Philadelphia: WB Saunders, 1994: Silverman LM, Christenson RH. Amino acids and proteins. In: Burtis CA, Ashwood ER, eds. Tietz textbook of clinical chemistry, 2nd ed., Philadelphia: WB Saunders, 1994: Astion ML, Orkand AR, Olsen GB, Pagliaro U, Wener MR. ANA-Tutor. A computer program that teaches the anti-nuclear antibody test. Lab Med 1993;24: Astion ML, Hutchinson KH, Ching AKY, Pagliaro U, Wener MH. Cytoplasmic Tutor: a personal computer program that uses high resolution digital images to teach the interpretation of a microscope-based laboratory test. M D Comput 1994;11: Wener MH, Pagliaro L, Orkand AR, Olsen GB, Astion ML. ANCA-Assistant. A computer program that teaches the interpretation of the microscope-based immunofluorescence assay for antibodies to neutrophil cytoplasmic antigens. M D Comput 1995;In press. 10. Cookson BT, Curtis JD, Orkand AR, Fritache TR, Pagliaro L, McGonagle L, Astion ML. Gram Stain Tutor: a personal computer program that teaches Gram stain interpretation. Lab Med 1994; 25: Ebell MH. Visual programming languages. MD Comput 1993;10: CLINICAL CHEMISTRY, Vol. 41, No. 9, 1995
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