October 2000

Greetings from the IHC Resource Group!

Yes, it has been a while, as some of you have reminded me recently. It’s good to know that you’re listening and that our work does not go unnoticed! We have some very exciting news, not the least of which is the establishment of the ISHRG Listserv by our own John McGinley, who will be the webmaster and moderator, along with Patsy Ruegg and myself. See “New Listserv” below for more details.

IHCRG and JOH...Perfect Together

I am very pleased and excited to announce plans for a column by the IHCRG in the Journal Of Histochemistry. Members of the IHCRG Committee are working with Dr. Jim Hendricks, Editor-in-Chief of JOH, and Rich Cartun, who is on the JOH advisory board, to decide on format and frequency. Stay tuned……!

E-mail Address Update

If any of you who received this bulletin by “snail mail” now has an e-mail address, by all means forward your e-mail address to the John McGinley at IHCRG@egroups.com. Online distrubution is easier, faster, and much more cost efficient!

Read on for more news and information!

Diana Goodwin

New Listserv

The NSH Immuohistochemistry Resource Group has a new listserv for distributing the bulletin and monthly newsbytes. Chairperson, Patsy Ruegg, approached me with a problem that she and Diana Goodwin had been confronting for quite a while. The problem was, how could the group maintain an updated e-mail distribution list, thus avoiding bounced e-mail messages? The answer was simple: create a listserv. If you are a member of the Histonet, then you are already familiar with listservs. The Histonet is an example of an unmoderated listserv, meaning that all members can post messages freely. The IHCRG listserv works in a similar fashion, but it is a moderated list, meaning all messages go through the listserv moderator before they can be posted. Currently, messages from group members are not being posted, only official bulletins and newsbytes. However, we are not opposed to the idea having an unmoderated listserv like the Histonet. Therefore, we would like to have your input on the following issue.

How should the new IHCRG listserv be used to best serve group members?

1. Moderated – distribution of official bulletins and newsbytes only. Members are not allowed to post messages.

2. Unmoderated – Allow members to post messages in addition to distributing official bulletins and newsbytes.

We welcome any and all feedback. Please e-mail your responses to IHCRG-owner@egroups.com . All current group members with valid e-mail addresses are automatically subscribed to this listserv. If you are a member of this group, but have not received an invitation to join the listserv, please send an e-mail request to IHCRG-owner@egroups.com . If you are currently subscribed to the list, but would like to change your e-mail address or make other changes to your account, please go to IHCRG page on the eGroups web site. http://www.egroups.com/group/IHCRG

John McGinley
IHCRG webmaster

NCCLS Guideline for IHC Testing Now Available  MM4-A, Quality Assurance for Immunocytochemistry; Approved Guideline Member Organizations $35 Nonmembers $85 This document provides consensus recommendations for the performance of immunocytochemical (also referred to as immunohistochemical) assays to promote a better understanding of the requirements, capabilities, and limitations of these diagnostic methods; to improve their intra- and inter laboratory reproducibility, and to improve their positive and negative predictive values in diagnosis of disease. MM4-A provides new updates on: ? Tissue collection and sample handling ? Fixation, antigen retrieval methods, and staining protocols ? Formats for reporting of test results ? Automated, rapid, and other new technologies Please contact Marc R. Schlank, M.T (ASCP), M.S. at the NCCLS Executive Offices for ordering information, and to learn more about participation on related projects (mschlank@nccls.org  or +610.688.0100 Ext. 123.) or you may order online by visiting the website at http://www.nccls.org

Workshop Review

Molecular Morphology Workshops

Santa Fe 2000

International Society for Analytical and Molecular Morphology
February 6-8, 2000 

A review presented by: Patsy Ruegg, HT(ASCP)QIHC
In 1999 I was awarded the Ventana IHC Scholarship by NSH. I used the $1000. awarded to go to the Molecular Morphology Workshops in Santa Fe, New Mexico. The meeting was held at the La Fonda Hotel, a beautiful historical hotel in downtown Santa Fe. The format of the meeting was a series of six half-day workshops. This is a rather small meeting with approximately 100 attendees. The workshops were hands on and very  personal. The equipment and technology display was awesome, something like I have never seen anywhere and certainly mostly nothing a routine histology lab could ever afford to buy. It sure was fun stuff to drool over, though.

Workshop #1: ISH & FISH

James F. Leary, Stefan Burde, and Susan M. Barns University of Texas Medical Branch, Galveston, Texas and Los Alamos National Laboratory, Los Alamos, New Mexico
This workshop began with an overview of basic FISH (fluorescence in-situ  hybridization) concepts including DNA:DNA, RNA:RNA, and RNA:DNA denaturation, hybridization and suppression-hybridization kinetics.  Probe construction labeling and amplification strategies for optimal signal-to-noise, sensitivity and quantitation were discussed. Application of ISH and FISH techniques were demonstrated by two examples. The first example involved detection of EBV (Epstein-Barr virus) DNA and mRNA sequences in human cells. The second example involved the use of FISH for correct identification of bacterial species.

Workshop #2 Laser Capture Microdissection (LCM) and the PixDell II System:
A New and Unique Tool for Rapid “Specific Cell” Molecular and Biochemical
Diagnostics by Dr. Jeffrey C. Travis ARCTURUS,Mountain View, CA 94043, U.S.A.

This system offered a means to isolate and extract an often small population of cells of interest from tissue sections. Gene expression and protein patterns require the cell capture and extraction of a microscopic subpopulation of homogeneous cells from their complex tissue environment. Laser Capture Microdissection (LCM) has been developed to provide a method for capturing and preserving specific cells from fixed tissue, cell cultures or cytological smears for subsequent molecular and biochemical analytical procedures. With this system and thermoplastic film is placed on the surface of a “fixed” specimen on a glass slide. The region of interest is visualized with an inverted microscope and an infrared laser (coupled to the microscope) is pulsed through the film. The film “melts” into the specimen and the result causes the specific cell(s) underneath to adhere to the film, with the morphology intact. The film (attached to a “cap”) is then lifted off the slide by a transport arm and placed in a standard microfuge tube containing extraction buffer for molecular and other analyses. We were shown this methodology and then given the opportunity to perform LCM ourselves. Image archiving allowed for photo-documentation of the entire process. This was pretty darn cool stuff, but very expensive equipment as you can imagine. The Prostate Cancer Research Lab at my institution actually has one of these instruments.

Workshop #3 IN SITU PCR: Detect Your Gene, and Gene Expression by the IN SITU PCR METHOD
Omar Bagasra, M.D., Ph.D., Lincoln University, PA 19352

Using a minute amount of DNA or RNA, and choosing a thermostable enzyme, one can amplify the nucleic acid of interest, which can then be analyzed or sequenced. This technique can be used to determine tumor burden before and after chemotherapy. In viral infections, one can determine the effects of therapy or putative anti-viral vaccination by evaluating the number of cells still infected with the viral agent, post-chemotherapy or vaccination. Amplification and detection of viral agents was presented and we were provided with the opportunity to perform PCR thermo-cycling and in situ gene amplification methods.

Workshop #4 INTERNET VIRTUAL MICROSCOPY
Thomas Jung, Product Manager/Director of Sales
Bacus Laboratories, Inc., Chicago, Illinois, USA

This workshop was the demonstration of this imaging system, which had unlimited uses for Anatomic Pathology to store, share, analyze, measure, enhance and report microscope images via the internet. Microscopic images could be reviewed using this system so that a pathologist would never have to go to the office. Images can be shared with colleagues for consult with a click of the mouse, making telepathology a breeze. Quanitative histomorphometry could be done using this system, although he was not able to show me this feature as it was not part of the package he brought to this meeting. This system uses a digital video camera looking through a microscope to capture the images. The image quality and manageability of this image system was remarkable, but once again, very pricey, beyond what most routine AP departments could ever afford.

Workshop #5 cDNA EXPRESSION ARRAYS
Megan R. Lerner, Jay S. Hanas, Eldon R. Jupe, and Daniel J. Brackett
Departments of surgery and Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Department of Veterans Affairs Medical Center; and Immunobiology and Cancer Program, Oklahoma Medical research Foundation, Oklahoma City, Oklahoma 73104
Natalie Luke, Ph.D., CLONTECH¸ Palo Alto, California, USA

This was the Hope Diamond of all workshops, which is why this equipment and methodology was bought and used in collaboration with so many departments, any one department could not afford this. This methodology took millions of dollars to just set up. cDNA arrays provides a method of performing comparative gene expression studies. This workshop concentrated on utilizing the Atlas cDNA expression Arrays from CLONTECH for expression profiling. Tissue handling, RNA purification, probe labeling, hybridization, washing and exposure of membranes was demonstrated and performed by use in this hands-on workshop. Computer software analyses of cDNA array data were presented. Over 100 gene expression sites could be demonstrated in one assay.

Workshop #6 AUTOMATED KINETIC MODE MOLECULAR MORPHOLOGY
Grigorios Totos, Abdelghani Tbakhi, James Pettay, Kimberly Christensen,
Comelia Hauser-Kronberger, Gerhard W. Hacker, Thomas Grogan,and Raymond Tubbs
Department of Clinical Patholgy, The Cleveland Clinic Foundation,
Cleveland, OH, The University of Arizona Department of Patholgoy,
Tucson, AZ; and the Institute of Pathological Anatomay, Salzburg Federal
Hosptial, Salzburg, Austria

This was the one workshop that I understood the most. An automated machine for doing insitu hybridization was introduced. This machine was developed and sold by Ventana Medical Systems International, Tucson, AZ. All the steps, including deparaffinization, cell conditioning, protease digestion, endogeneous peroxidase blocking, denaturaztion, hybridization with biotinylated genomic probe, stringency washes and all signal detection steps were done on this machine (Discovery ISH instrument). Each of the 20 slide stations was individually temperature controlled. We demonstrated HPV (human papillomavirus) in human cervical carcinoma cell lines, cytologic and histologic preparations, during a 5-hour automated procedure. This is a machine that I could and would use in my lab, but unfortunately, it too costs a whole lot more than we could ever afford. I’ll keep it on my wish list.

This 3-day meeting was a real learning experience for me. The location and hotel are beautiful, and Santa Fe has always been one of my favorite places to visit. I was in awe of the technology and equipment presented at this meeting. In most cases only the very rich pharmaceutical companies or very large generously funded research institutions could afford this technology. The Laser Capture Microdissection (LCM) methods were developed through a Cooperative Research and Development Agreement (CRADA) between the NIH and Arcturus Engineering. This says to me that NIH was probably the brains behind this technology, and has enlisted Arcturus to manufacture and market it. I would definitely attend this meeting again if I were given the opportunity. I have joined the International Society for Analytical and Molecular Morphology and will try to keep up on their doings. –PR

Awards

Two of our group’s members were honored with awards at this year’s NSH Banquet on September 17th at Milwaukee’s beautiful Midwest Convention Center. Congratulations to Patsy Ruegg, who is this year’s recipient of the NSH Foreign Travel Award, sponsored by SurgiPath. In May, 2001, Patsy will be traveling to England to study immunohistochemistry on plastic embedded tissues with Neil Hand at the University of Nottingham. And also to Diana Goodwin, who received the Richard Allan Scientific Continuing Education Scholarship. Diana will use her scholarship to continue her undergraduate studies at Widener University in Chester, PA.

THE END

Have a safe and Happy Halloween!