As the Wheel T...Tampa, FL [email protected] Dreama Hall Continued Education Chairperson Largo, FL 33771 [email protected] Nisha Crosby Newsletter Editor Apopka, FL [email protected]

Message from the PresidentI am very excited to have this opportunity to write to you all as the new President of FSH for 2014-2016. I want to thank Jerry Santiago, immediate Past President, for all his support, encouragement, and guidance over the past 5 months. Coming right out the gate, I was given the task to begin the search for a new location for our 2015 annual meeting. That decision was made easy by the wonderful location that we were at this past year. It is my pleasure to announce that our meeting will once again be held in Orlando at the Buena Vista Palace Hotel & Spa on May 14-17, 2015. I would like

to take this opportunity to thank all our vendors for their support of our annual meeting as well as the way they support us all year long. We would not be able to do the things we do without their assistance.

Additionally, I would like to thank my Board for all their support and suggestions, and I look for-ward to getting to know each one better throughout the year as we work as a team. We have added a few new people to the Board this year. I want to welcome Betsy Woessner as our Finance Chair and Nisha Crosby as our Newsletter Chair and Editor. We would very much like increased participation from the membership. If interested, look at the list of committees and see which one you might like to get involved with and contact the chairperson who oversees that area.

We have some membership items that we are looking to address over the next few months so that they can be presented for voting at our general membership meeting held during our annual meet-ing. We have already put in place free 2 year membership, renewed annually, for students in a NAA-CLS approved program and the acceptance of membership will require verification by the Course Program Directors. We are also looking to make our membership year coincide with our annual meeting. The benefit will be that all money will be collected at one time and budgeting should be made easier. As members, you would automatically get the membership rate for the meeting and registration. Since all changes need to be voted on at our general membership meeting as per our By-Laws, it will be presented that we utilize the available electronic technology (such as the one used for bi-annual voting) or similar thereof for the passage of rule changes. This will enable us and future Boards to streamline changes and put the changes in place more quickly.

As I settle in to my new position, I want to thank our membership for their support. I also would like to mention that we are in a renewal cycle for FSH Membership at this time and would appreci-ate your support by joining or renewing your membership with the society. I want to assure you that we as a Board will continue to build on the strong foundation of this great society as we explore new opportunities and continue to move our craft to professional status like the professionals we already are.

Let’s enjoy the next two years together!

Kind regards!

John Shelley

In This Issue

Florida Society for Histotechnology

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Volume XXXVIII

Board of 2Directors

2015 Annual Meeting 3

2014 Meeting 4-5

Membership 6

NSH Corner 7

NAACLS 8 Immunohistochemistry 10-12

CEU Questions 14

Fall Edition, 2014

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Board ofDirectors 2014-2016

Board MembersJohn ShelleyPresidentKissimmee, [email protected]

Michelle FosterVice PresidentRuskin, FL [email protected]

Alice BonaiutoSecretaryBradenton, [email protected]

Loretta SaylesTreasurerIndialantic, [email protected]

Jerry SantiagoImmediate Past PresidentOrange Park, FL [email protected]

Committee ChairsAmy ItalianoAwards/Scholarships/ElectionsTampa, [email protected]

Dreama HallContinued Education ChairpersonLargo, FL [email protected]

Nisha CrosbyNewsletter EditorApopka, FL [email protected]

Linda BlackburnHistorianSt. Petersburg, FL [email protected]

Dreama HallRegion I CoordinatorLargo, [email protected]

Bonnie CohenRegion II CoordinatorMiami, [email protected]

Charlotte KopczynskiMembership ChairpersonPalm Harbor, [email protected]

Betsy WoessnerFinancial ChairpersonOrlando FL [email protected]

Michelle FosterBylaws ChairpersonRuskin, FL [email protected]

Michelle FosterEvent Planner/CoordinatorRuskin, [email protected]

As the Wheel Turns Volume XXXVIII

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2015 AnnualMeeting

Save the DateFSH Annual Meeting 2015

May 14-17, 2015

Buena Vista Palace Hotel and Spa

buenavistapalace.com

Soaring 27 stories above the world’s favorite destination, Buena Vista Palace Hotel and Spa offers a premier meeting

experience in the heart of Downtown Disney.


2014 Annual MeetingAward Recipients

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We are thrilled to congratulate all of our 2014 Annual Award Recipients!

Histotechnologist of the Year Tatiana Podrabinek, HT (ASCP)This award is reserved for a technologist or technician who has been dedicated to the advancement of Histotechnology as a profession and has contributed to the field of Histology. The recipient of this award received a Histologist Sculpture courtesy of C.L. Sturkey and a $250 grant from Newcomers Supply.

Vendor Appreciation Award Carlos Cuello from C.L. SturkeyThis award recognizes a sales representative, regional manager, corporate owner, technical representative or corporation that has supported the Florida Society for Histotechnology and has promoted and participated in the field of Histotechnology.

Pathologist of the Year Farah Khalil, M.DThis award recognizes a Pathologist who has made a significant contribution to the Florida Society for Histotechnology through their support of the profession of Histology. This person is one who has been an outstanding mentor to the Histotechnologist(s) in his or her laboratory. This person treats all hospital personnel with respect and always has the interest of the patient in mind.

Career Achievement Award Michelle Foster, HT (ASCP)This award is to recognize an individual who has made a significant contribution to the Florida Society for Histotechnology through their dedication and service to the profession of Histology. This person is one who has outstanding leadership qualities, inspires others to pursue their love of our field, and who continually makes technical and academic progress within their place of employment.

Bobbie Spillan Award Nancy Shellhorn, HT (ASCP)This award is given in memorium of Bobbie Spillan, the first President of the Florida Society for Histotechnology, and is given to a de-serving Histotechnologist, who has achieved excellence in the field and supports continuing education.

President Choice Award Lucy Soto, HTL (ASCP)For unconditional support of the President of the Florida Society for Histotechnology

Excellence in Histotechnology Scholarship Anilec LlanesSponsored by Poly Scientific

Research & Development Scholarship Ebone MartinSponsored by Poly Scientific and Dr. José V. Suarez-Hoyos

Suarez-Hoyos Scholarship Julie AyoSponsored by Dr. José V. Suarez-Hoyos


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2014 Annual MeetingPhotos

2014 Annual MeetingCharity GivingOur charity for the 2014 Annual Meetings was Hub-bard House, a “home-away-from-home” for the families of patients living outside the Central Flor-ida area. This year we were able to donate everyday items Hubbard House could use for these families on a daily basis. In addition, we were able to also give to them over $200.00 in cash and gift cards to help with their $500.00 a month budget for operating the House. The House is sustained by the hard work of Peggy Smith, along with her staff and volunteers, and her ability to get donations from places like Second Harvest Food Bank and Disney, to name a few. We are sure these donations went on to play a vital role in the incredible work Hubbard House provides to these families.

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NSH Corner

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I had the opportunity to go to the NSH Symposium and Conven-tion in August at the Convention Center in Austin, Texas. Austin is known for being the “Live Music Capital of the World”, and I would not argue with that claim.

As I walked the streets in the evening you would see roof top ven-ues with all types of music giving an eclectic mixture of rhythms and sound. What really caught my fancy was the Tex-Mex flair in the food choices and the barbeque that was available on just about every street corner. This was beyond what was actually taking place in the Convention center. The staff of NSH should be commended for another fantastic meeting. With nearly 2,000 people and over 120 classes over the 5 day event, if you did not walk away from the event recharged and excited for the work that we perform on a daily basis then you need your pulse checked. The awards banquet was well done and the Texas Society did a great job with providing the entertainment after the awards and meal was served.

At the general membership meeting, the Florida Society was given the award for best website for the third time. We are appreciative of the job that Christina Kranson does to maintain our website. You may have already received the survey from an outside consulting firm for NSH. This survey is important for knowing what the general membership needs are at this time and for the future and how to better serve these needs. If you did not take part this time, please make every effort to become more involved with responding in the future. The NSH is a great tool for us as we continue to morph from our routine processes to the more advanced and sophisticated molecular ones.

John Shelley2014-2016 FSH President

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Recent Change to NAACLS Standards


At its September 2014 meeting, after reviewing over 100 public comments, the NAACLS Board of Directors ap-proved a change to Standard I.D.1.g, requiring HT programs to culminate in either an associate degree or higher, or a certificate for students who hold or complete the required degree (see below). Remaining consistent with similar changes in the past (notably in 2001 with MLT programs), NAACLS accredited HT programs will have until their next accreditation review to show proof of compliance with this standard. If an accredited HT certificate-based program has an accreditation review coming soon and needs more time to work to be in compliance with this Stan-dard, the program is encouraged to contact NAACLS staff in order to discuss review scheduling and extension op-tions.

The Board of Directors also viewed public comments related to a proposed change to Phlebotomy Unique Standard VII.A.1.b, and it was decided that a clarification would be added to the NAACLS Standards Compliance Guide to include the ASCP-BOC as a recognized agency for the certification qualification for program directors, leaving the current Standard language intact.

Please contact NAACLS if you have any questions on these changes!

Approved Change to Standard I.D.1.g:

1. Sponsorship

2. Responsibilities of the Sponsor

A. The sponsor has primary responsibility for

1. Ensuring that graduates of the program have obtained or will obtain the minimum degree and/ or certificate upon completion of the program.

MLT and HT programs: an associate degree or higher, or a certificate for students who hold or complete the re-quired degree.

Edward RotchfordAccreditation Specialist/Volunteer Services Manager

Reference:Rotchford, Edward. (2014). Recent Change to NAACLS Standards. Retrieved from http://www.nsh.org/content/recent-change-naacls-standards

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Getting the Most Out of Your ImmunohistochemistryTechnically immunohistochemistry is relatively simple and straightforward. However, there are a lot of vari-ables that must be identified and optimized for each study. Immunohistochemistry may also involve trou-bleshooting of a variety of factors.

I am about doing things right the first time. A simple, but sometimes over looked step is to choose antibod-ies that work for immunohistochemistry. This can save a lot of headaches down the road. Search for specific antibodies from literature, vendors as well as your peers.

There are several things to keep in mind with antibod-ies. What might work well in one laboratory might not be optimal for your laboratory. Each antibody needs to be tested with your staining system. Antibodies over time can lose their staining intensity. Exposure to air and light can cause this.

Sometimes we overlook the importance of our controls. Date and number each batch of controls that you cut. Keep track of who cut what and when. Tissue sections kept at room temperature for more than a few weeks show loss of some epitopes. Some antibodies are more sensitive to this than others.

When we get into troubleshooting, it is important to set objectives to determine a goal for each run. Change one variable at a time such as incubation time of primary or detection system or the pretreatment method used. It is also very important to document your results. If optimization is not successful or if problems with op-timized antibodies arise, troubleshooting may be neces-sary to resolve the staining issues. Two main problems are common with IHC staining: Weak or no staining as well as non-specific staining and/or background.

One of the first things to check with weak or no stain-ing is to check the primary antibody. It may have been omitted by mistake. This is especially true if you are

making dilutions. It also may be defective or out of date. Check the expiration date of the antibody. It also may be necessary to use a new lot or batch. Antibodies lose potency over time and may require higher concen-trations. As we get busy in laboratories, mistakes can happen. Sometimes we need to recheck our calcula-tions to make sure that we did our dilution correctly. Do not overlook the importance of the diluents that we use. Some may contain substances that interfere with staining. Sometimes we may be dealing with inactiva-tion of the antibody. I found this to happen most often when I had freeze thaw patterns with the antibody. This typically happens if we freeze small aliquots of certain antibodies. Wrong incubation times or temperatures may also need to be addressed.

Antigen retrieval can also play a role with weak or no staining. The method that we have chosen may be in-appropriate. Some of our instruments allow us to in-crease our times in the retrieval. You can also look at the other retrievals that are available to you. Also keep in mind that some antibodies do not require antigen re-trieval. Check pH before use and also ensure that our concentration calculations are correct. We need to be sure that our temperatures and incubation times for our retrieval are correct. Do not rule out the opportunity for dual retrieval for difficult antibodies. Heat and en-zyme retrieval can work well together. Start out with a ratio of ten minutes in heat and five minutes in enzyme or vice versa. Tenascin C is an antibody that comes to mind for this. If all else fails, refer to the specification sheet for the antibody you are working with.

Not using the appropriate control can play a role in weak or no staining. Sometimes when you purchase control slides, animal tissue may be used. You may also have a control that has low expression level. Keep in mind that you may have cut past the area of interest. Take charge of your controls and start looking for drop off before this becomes a major problem.


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Ancillary reagents also play a role in weak or no stain-ing. Incorrect order of steps can be a problem. Dis-tilled water pH is a problem that I encounter in the field. We need to be diligent in keep track of the pH of our water. Distilled water can contain inhibitors of peroxidase reaction. Again, some antibodies are more sensitive to others with this phenomenon. Also when you troubleshoot the water, do not forget the buffer. Distilled water is often times used to dilute the buffer for your final working solution. The buffer may also be contaminated or out of date. Also check your de-tection kit for the same reasons.

As we continue to discuss weak or no staining issues, we need to turn to the protocol itself. Incubation times in our protocols can be too short. This also applies to our retrieval protocols. We are taught that washing is a very good thing. Keep in mind that our washes need to be stringent but brief. You can cause dissocia-tion of antigen-antibody complexes by too extensive or vigorous washing. Also watch the washes with red detection kits. We can completely wipe out our stain-ing or cause bleeding that we sometimes see. When we look at our protocols, we have to think outside the box. The peroxidase block can inhibit staining based on where it is in the protocol. Moving the peroxi-dase step down in the protocol can enhance staining. PAX5 tends to work better when the peroxidase block is moved from the beginning of the protocol to after the polymer step. Too many blocking steps can also inhibit staining.

Mounting media is another variable that should not be overlooked with weak or no staining. Choose the correct mounting media for the utility that you are us-ing. This is also something to consider when working with red detection kits. Some enzymatic products are soluble in alcohol, xylene or other solvents.

Over staining is another common problem that we

see. Again, we need to start with the protocol itself. Incubation times of our protocols, as well as antigen retrieval, may need to be shortened. If you are work-ing with concentrates, a dilution study may be needed to determine the correct concentration for your pri-mary antibody. Sometimes the incubation tempera-ture for your primary antibodies or antigen retrieval is too high. The substrate incubation times may also need to be adjusted.

The main cause of non-specific background staining is non-immunological binding of the specific immune sera by hydrophobic and electrostatic forces to cer-tain sites within tissue sections. These hydrophobic interactions can be decreased by formulation of the diluent buffer. This can decrease the buffers ionic strength. Tween 20 detergents as well as a 1% bovine serum albumin can also be added to the diluent to reduce non-specific binding due to the hydrophobic interactions.

When we experience non-specific or background staining the blocking step must be considered. Ensure blocking solution is aligned with species of antibody used. Increase the concentration of our blocking so-lution until the desire results are achieved. Increase incubation times as necessary.

High background can also be caused by too strong of concentration of your primary antibody. Non-specific binding can also arise from our secondary an-tibodies. You can also experience problems when you choose an antibody that is made in the same species as the animal that you are testing.

We also need to review our antigen retrieval when we experience high background or non-specific staining. Over retrieval is often the problem. This can be ad-justed by decreasing our retrieval times or tempera-tures. We can also choose a lower pH retrieval. Over

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Getting the Most Out of Your Immunohistochemistry

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retrieval is often the problem. This can be adjusted by decreasing our retrieval times or temperatures. We can also choose a lower pH retrieval. Over digestion can also cause this problem. Look at your enzyme retrieval time, temperature and concentration.

The protocol may also be adjusted for non-specific or background staining. We mentioned the pitfalls with over washing. The converse is also true here. If we do not wash adequately, some of these issues may occur. Do not let your slides dry out!

Sometimes it can be very frustrating when tissues come off of our slides. One of the causes is inadequate drying of the slides after cutting them. Also be mindful of too aggressive of antigen retrieval. Do not forget enzyme retrieval here. Do not rule out a bad lot of charged slides. I have experienced this myself.

Brown/black pigment is something that we sometimes see on our slides. Make sure that proper maintenance is done on our instruments. Also check the DAB itself. Sometimes we may also see this from the polymer step in our detection protocols. I have also seen this from a lead pencil when writing on the tops of the slides.

Hopefully these tips will help you out. I know there seems to be a lot that can go wrong, but with a little persever-ance, you will get a nice result. Make sure that you find a protocol that is right for your laboratory and remember, troubleshooting is everyone’s job.

Steven Westra

Antibody Staining Consultant

Leica Biosystems


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CEUQuestions


Instructions: Circle the BEST answer.

1. Antibodies over time can lose their: a. pH b. Charge c. Staining intensity d. Epitopes

2. Control tissue sections over time can show loss of: a. Tissue b. Antibodies c. Color d. Epitopes

3. Variables should be changed _______ at a time. a. One b. Two c. Three d. Four

4. What are two common problems in immunohisto chemistry? a. Weak/no staining and specific staining b. Weak/no staining and non-specific and/or background staining c. Over staining and specific staining d. Over staining and non-specific and/or background staining

5. Heat and enzyme retrieval do not work well to-gether. a. True b. False

6. The main cause of non-specific background stain-ing is non-immunological binding of specific immune sera by hydrophobic and electrostatic forces to certain sites within tissue sections. a. True b. False

7. Non-specific binding can occur when the antibody is produced in the ______ species as the animal being tested. a. Opposite b. Same

8. What are some reasons for loss of tissue on charged slides? a. Inadequate drying b. Aggressive antigen retrieval c. Bad batch of charged slides d. All of the above

9. Factors that can cause weak or no staining: a. Primary antibody b. Antigen retrieval c. Inappropriate Control d. Order of ancillary reagents e. All of the above

10. Troubleshooting is everyone’s job? a. True b. False

Cost: FSH Member – FREE; Non-Member - $10.00Mail your answers and applicable fees to:Dreama HallFSH CEU Chairperson1155 Lake Avenue, S.E.Largo, FL 33771


Florida

Soci

ety for Histotechnology •

FSHEst. 1973

Nominate a deserving co-worker for an award.

Nominate a deserving student for a $500 scholarship to help them succeed.

Did you know?You can renew your membership online! www.fshgroup.org

We want to hear from you. What’s new in your lab? Do you have an opinion? Send us an article you feel would be of benefit to your fellow Histotechnologists. Do you have a question, a tip or a comment? It’s easy, just contact the FSH newspaper editor for consideration.

It’s time to renew your FSH membership! We want you to be a mem-ber of the only professional organization for histology professionals in Florida. As a member, you will receive:

• E-Newsletter posted on the FSH Website• Continuing education opportunities at regional and state meetings at a dis-

counted rate. • Self study continuing education opportunities through our website offerings• Access to updated profession information on the FSH website at www.fsh-

group.org, including links to NSH, ASCP, and Clinical Lab Board• Continuing Education Credits provided by FSH free of charge• Representation at the Board of Clinical Laboratory meetings• Representation in the House of Delegates during the National Society for

Histotechnology Symposium/Convention• Updates from the NSH Regional Director

The annual membership dues are $35.00 and membership runs from September 1 to August 31. To renew, please visit www.FSHgroup.org.


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Documents

As the Wheel T...Tampa, FL [email protected] Dreama Hall Continued Education Chairperson Largo, FL 33771 [email protected] Nisha Crosby Newsletter Editor Apopka, FL [email protected]