Category Archives: Skills

A detailed description for all of my skills

Tissue Micro-Array TMA

01/29/2016 Eric Hanson Leave a comment

This is easily the most complicated process performed in the immunohistochemistry (IHC) lab at Caris Lifesciences. All of the quality control that is done in the lab is dependent on the TMA. So without these cutting and staining well, the lab comes to a standstill and repeats have to be performed on a large number of tests. This delays production and costs enormous amounts of money. The TMA production was my primary responsibility at Caris. I also developed a method to save the company between 100-350k on bulk tissue costs by implementing a shallower tissue punch in the block.

Awarded after my TMA workflow processes changes were implemented

I can’t go into too fine of detail on the process, but I’m quite familiar with how these are made. I’ve written the complete standard operating procedure (SOP) on this process, and many other processes, for Caris Lifesciences.

The outline of the process for making TMAs is as follows: bulk tissue purchasing, bulk tissue cutting, quality analysis/positive staining, block marking, TMA blank block production, tissue punching, and lastly, quality control.

Bulk Tissue Purchasing
It’s incredibly hard to find a reliable source(s) for tissue and the distribution/purchasing of such tissue is sort of in a legally gray area. The tissue we receive must stain positive, dependent upon tissue type, for the immunohistochemical (IHC) stains we are performing on patient tissue. So this means we need human tissue that has tumor in it. Thankfully the process we have implemented uses the least amount of tissue as possible to maximize the usability of the bulk tissue blocks.

Bulk Tissue Cutting
After the bulk tissue blocks are received, each has to be sorted into tissue types and then the tissue types that are going to be punched in to the TMA have to be cut on the microtome. One section for each stain it is suppose to stain positive for must be cut. For some of the tissue types such as HER2 positive breast tissue, only needed one slide cut, but for others, as many as 11 slides per block had to be cut.

Bulk Tissue Quality Analysis
Each block has to stain positive for each of the stains it is going to be used as a positive control on. So for each block, the IHC slides must be looked at closely to make sure uniform staining has occurred and it is staining the right sections of tissue. These IHC tests usually stain a certain tissue type the best such as epethilial, nerve, capillary wall, mucousal, etc cells. During the tissue validation phase, all the different tissue types are stained with all of our stains and then a pathologist looks at them to determine which ones they want for positive controls.

microtomy — Sections laid out on a warm water bath

As part of the quality analysis of the tissue, once determined that that tissue block is suitable for use in the TMA, the stained slides are then each marked to highlight the area we are interested in punching. These punches go into the blank TMA blocks to make the cutable TMA.

Block Marking
After we’ve determined that the block has stained positive for all of the tests it needs to, the positive portions are marked on the coverslip of the glass slide. These marked slides are then overlaid on the block and used to mark the tissue in the block with a statmark pen.

TMA Blank Block Production
Now that there are a lot of blocks marked of each tissue type that is going to go into the TMA, we need to make TMA blank blocks to put the punched tissue into. Depending on the size of the punches and the number of punches for the TMA, a mold size is chosen. Plastic cassettes, generally of a different color than the patient specimen color, are printed out and placed on top of each mold. Paraffin is poured in the mold and then the perfect amount of paraffin needs to remain in the back of the plastic cassette. Without the paraffin backing the cassette, it’s really easy for the portion in the mold to fall off when cutting the cassette or punching through the plastic cassette when making the TMAs.

Tissue Punching

During this step a predefined set of coordinates is used to punch certain tissue types into certain areas of the block. To streamline the punching process, I would set the instrument to the first coordinate and recipient-punch all the blank TMA blocks at that location. Line those up, usually 10 at a time, and then use the bulk tissue blocks and donor-punch them set aside the cores on a cutting board. To prepare the cores for the blank TMA blocks, the paraffin on the bottom has to be sliced off using a scalpel. The tissue is then stacked in the hole on the donor block (blank TMA block). The whole process then repeats itself for each coordinate.

Quality Control of the Finished TMA Block
Once a block has been fully completed, through trial and error, we figured out that baking the blocks for a set amount of time helped embed the tissue cores in the paraffin a little better. Most TMA blocks have problems with the tissue cores being pulled out by the blade during microtomy.

Once they are cooled a couple sections are cut off each and they are stained H&E to look for the correct cell morphology in each core, and then put them into production. Each microtomist gets a block and uses it to cut a section on each slide that already contains patient tissue and that is also going to be stained one of the IHC stains we did at Caris.

Skills

Tissue Embedding

01/28/2016 Eric Hanson Leave a comment

Tissue embedding is a pretty simple task. Processed tissue is placed in a mold and paraffin is then drizzled on top of it. A plastic cassette is then placed on top of the mold and it’s set on a cold plate to cool. This creates the formalin fixed paraffin embedded (FFPE) block. These are the common standard used in all of histology. If a portion of tissue is too large for biggest mold, gross dissection of the sample is required.

This was one of my tasks when I would come into the lab on the weekend to gross the saturday samples. Sometimes we would receive samples that were already tissue processed, but received in a very large cassette, or stuffed with too much tissue for the mold they used. In this case, the FFPE block would be melted down and the tissue sectioned so it fit into one of our molds.

When performing microtomy, sometimes the tissue would pop out of the block, crack, have bad paraffin quality, or any number of things that inhibited cutting. The block would then be re-embedded using our embedding station, logged, and then sectioned with better results. This was also necessary if the tissue was on an odd plane, or one portion of the tissue was embedded further into the paraffin than the section that is hitting the microtome blade.

tissue embedding — Tissue-Tek embedding center that I’m familiar with

When I was making Tissue Micro Arrays (TMA), I would make 100 blocks at a time using this station. It was quite tedious, and getting the paraffin to properly fill the back of the plastic cassette was hard. It is necessary to get enough paraffin the back to avoid over punching the block or having the paraffin break off while performing microtomy.

Skills

Slides: Organization Distribution and Filing

01/28/2016 Eric Hanson Leave a comment

An often overlooked and very necessary part of the lab is storing specimens. This was the job of the laboratory aides in the lab that I worked in. It takes a tremendous amount of time to make an efficient system for dealing with the flow of specimens we received while working at the lab at Caris Lifesciences.

The organization flow started when the specimen entered the company where it was given a case number. This number was used for all of the logging that occurred when the specimen passed through all the steps up to the case report. At any time, we could track where a case was based on the fastidious logging at each station. Because the life of someone is on the line, these logging steps prevent the mix up of tissue from one patient to the next. There are also a lot of auditing that goes on to make sure everything was where it should be. The laboratory information system (LIS) and sharepoint were some of the tools used for data logging and mining.

slide organization distribution filing — Slides filed by case number in a temporary cardboard container

The lab aide spent most of their time moving slides around the laboratory. This distribution is key to efficient turn around time (TAT). We prided ourselves at Caris, that we could get a specimen in, and provide all the work required to make a good therapy regime, with a window of only 7 days or less. If all of the machines were operating and there were no holdups, this happened most of the time. Sometimes machines would go down or an antibody would not work properly, which would really slow the process down. At this point, the organization and distribution of the slides are key to making sure that everything gets done in the order it is suppose to.

Skills

Quality Analysis and Quality Control of IHC Stains

01/28/2016 Eric Hanson Leave a comment

At Caris Lifesciences, all of the stained slides went through a quality control process before they were able to leave the laboratory. Once out of the lab, they were looked at by a pathologist who put together a recommended treatment plan for rare and hard to treat cancers. They used a combination of immunohistochemical (IHC) stained slides, a molecular profile, and a comprehensive database of patient treatment. All of this was done in house, which helped Caris shine from the rest of the theragnostic labs.

As I progressed as a histotech, I learned more and more about looking at slides and stains to determine if things stained properly. I received training from pathologists, coworkers, and supervisors. If I had a question on a stain, I would always get the best answer I could from a supervisor. It was painstaking to learn it all, but it really helped me develop and understand and knowledge base of the stains we performed and also what a lot of tissue looks like under a microscope.

quality control and analysis using H&E — This is a hematoxylin and eosin, or H&E stained slide. These are the bread and butter of histology. Most diagnostics start here. This is a section of colon.

The majority of what I learned was developed when I was making tissue micro-arrays (TMA). Please see the section I’ve devoted to TMAs.

quality control and analysis TMA — This is a very large tissue micro-array

Quality control for the automated stainers (Ventana and Dako) consisted of looking at the TMA section placed on each slide. These act as positive controls for the stains. Several different tissue types were present that were know to stain positive for all of the stains. I was in charge of selecting tissue and passing the tissue by sectioning and staining each tissue type for all of the stains it was supposed to stain positive for. This was step one of the quality control process.

quality control and analysis HER2 — This is a HER2 positive stain on breast tissue. It’s one of the prettiest stains resembling a stained glass window.

The final step before the slides were delivered to a pathologist was the QC stage. Someone would sit and look at the TMAs on each slide and also the tissue to make sure that it didn’t fall off and had a uniform stain. Sometimes in microtomy a section can be placed on a slide that is thick on one side and thin on the other, or had chatter or other anomalies. Once this was done, the case was logged and sent out of the lab. I spent a lot of days at the QC station. It was always a nice change of pace from the frustrating microtomy or monotonous staining procedures. It was fascinating to see all the different types of tumors and stains.

Skills

Polymerase Chain Reaction

01/27/2016 Eric Hanson Leave a comment

When I was attending college we practiced this process in the laboratory. I thought it was quite intriguing the way the polymerase could just pick up floating genetic material and amplify the DNA through a series of heating a cooling steps.

I’ve never had the opportunity to employ it in a work setting, but we did quite a bit of it when I was in the molecular biology laboratory and also when we were extracting liverwort DNA.

Skills

Microtomy: Sectioning Tissue at 4 micron

01/27/2016 Eric Hanson Leave a comment

The staple of the histology lab is microtomy of formalin fixed paraffin embedded (FFPE) blocks. The microtomes and our process at Caris Lifesciences were all validated for 4μ thick sections, so all of my experience is cutting sections that thin. Frozen microtomy uses thicker sections, and some test require thicker sections to work, but we’ve found that 4μ are about one cell thickness. This allows for great visual representation of protein expression in cancer cells after our IHC staining process.

Microtomy can easily be the most frustrating thing to do in the laboratory. It only becomes so if the tissue is processed incorrectly, which a lot of the tissue I’ve sectioned has been poorly processed. There is a special window of just enough water in the tissue. Too much and it cuts poorly, and too little it just turns into powder or hardens to the point of ruining blades. Improperly decalcified bone specimens are also a bear to cut. Usually you just get what you can with those.

The process we used when doing microtomy is a bath full of heated water and ice blocks. The block goes on the ice first and then the microtome. The microtome chuck is then lined up to the face of the paraffin block and the first initial sections are cut. This is called facing the block. Depending on how that went, you would then either set the block back on the ice, or in the warm water bath.

A ribbon formed on a microtome which is then moved to a warm water bath and then each section is picked up on a glass slide

After the initial facing of the block, you try to get a good ribbon going where each slice connects to the other due to the friction of the tissue and paraffin sliding off the blade. This ribbon is transported very careful to the warm water bath where the paraffin becomes clear. Each segment of the ribbon is teased apart using forceps, and then gently moved and picked up with a glass slide. The slides then go in a drying rack and then into ovens to bake the tissue on the slides.

Once the slides are baked, the tissue has adhered quite well to positively charged glass slides. At this point, the tissue is ready for all the testing it’s going to be enduring. Some processes require a nuclear fast red to make the tumor area pop out, and then the tumor is scraped off the slide under a dissection scope. The powder then runs through an assay to get the DNA/RNA into well plates for molecular analysis. The IHC lab I worked in ran a lot of IHC tests on each case. All of this work would be impossible without the use of microtomy.

I’m familiar with the Leica 2235 and 2255 microtomes. The first is a manual microtome, and the second is an automated one with a foot pedal. I have yet to see anyone use the automation feature on those microtomes. I don’t prefer one over the other, they both cut excellently.

Skills

Microscope: Compound and Dissecting

01/27/2016 Eric Hanson Leave a comment

I’ve spent a lot of time in front of a microscope either chasing around a micro invertebrate to analyzing a histological stain for Q/A. During lab courses in school I was always the one person that was good at following around single celled organisms so everyone could get a glimpse at it.

When working at the Desert Studies Center, Zzyzx, CA on the Mohave Tui Chub project, I spent huge amounts of time in front of a stereo dissecting microscope counting zooplankton. It was very tedious work, but a really fun way to explore a world invisible to the naked eye.

At Caris Lifesciences, working in the immunohistochemistry (IHC) lab required immense amounts of microscopy work. We had really nice stereo compound microscopes that every single slide went through. When I was making tissue micro-arrays (TMA) I had to quality control (QC) all kinds of tissue to make sure they would work for use in the TMA. I also spent a lot of time looking at each of our IHC stains to make sure they stained properly and the stainer machines were still staining with the intensity we were looking for. In order for a slide to leave the lab and get in front of a pathologist, it had to go through our internal quality analysis (Q/A) team.

Skills

Laboratory Information System (LIS)

01/26/2016 Eric Hanson Leave a comment

The laboratory information system (LIS) is a system used in a laboratory to track everything that happens in a lab. Most companies use a combination of paper and electronic logs to track everything from specimen arrival to machine maintenance. Storing the information of what happened to a specimen as it goes through the lab is not only essential, but are required by most laboratory accreditation organizations.

While I worked for Caris Lifesciences, every single thing we did in the lab was tracked via LIS, MS Sharepoint, or by pen and paper logs. This was key to being able to find cases if there were problems at any point during the process. Dutiful use of these systems provide many audit points to make sure nothing is lost in the process, and if something does go missing, this provides a great starting point to finding it. There are also many points during the process that mistakes in labeling or specimen handling can be caught before they become a problem.

Another product of using a system that tracks everything is that you get to run statistics on how the process is working. This can allow further refinements to streamline efficiency. With all that data at your disposal significant findings can be found by data mining.

Skills

Gross Dissection

01/26/2016 Eric Hanson Leave a comment

Gross dissection covers a wide variety of tasks that are performed in medical labs which ranges from removal of surgical samples from a patient to slicing up a needle core biopsy. When I was working for an oncology lab, the dissection I routinely performed was on small surgical samples that arrived in formalin.

The lab I worked in received samples from all over the world. Sometimes they came from a hospital that had a tissue processor and an embedding station. These tissue samples were already in a paraffin cassette and required nothing further in preparation of microtomy. Some of the odder samples we received were processed tissue in a square block of paraffin, large pre-cut tissue (for larger plastic cassettes), and whole organs or huge amounts of fatty tissue in large formalin vials.

We received tissue in this style of vial filled with 30:1 formalin to tissue ratio

Gross dissection is really the first thing that needs to occur to a tissue sample before we can process it through the lab. Without the tissue going through a tissue processor, which removes most of the water out of the sample, we can not cut it up or run any tests on it. So, I volunteered to be the person that came in on the weekends to gross the surgical samples we received.

The first step is looking at the pathology report to determine what part of the body the tissue came from. This information may help us determine how to section the tissue if it is too large to fit in one of our paraffin molds. The next step is measuring the dimensions of the tissue and determining where the tumor is. The major idea behind gross dissection for our purposes, is to expose the largest area possible so we have a lot of tissue to look over during the immunohistochemical (IHC) tests. The last step is determining if it needs to be bisected, trisected, loafed, or many other forms of further cutting.

The tissue goes in the center of the plastic cassette and the lid is then closed to prepare the sample for tissue processing

During the measuring and cutting process, we had to take extremely detailed notes on what color the tissue was, it’s dimensions, all the personal identifying writing on the formalin vial, and how it was cut and placed in x number of plastic cassettes. Once those notes were good, we would place all of our tissue cassettes into a tissue processor, dry out the tissue, and then embed it in paraffin using the right sized mold for the tissue. Most modern day labs all use formalin fixed paraffin embedded (FFPE) blocks for microtomy. It’s really the industry standard, and it is what I have the most experience with.

This is what a typical plastic cassette with processed tissue embedded in paraffin looks like

Skills

Gel Electrophoresis

01/25/2016 Eric Hanson Leave a comment

I took a molecular biology course when I was attending Minnesota State University: Moorhead (MSUM). It was the first course I took that required extensive laboratory time. I was a great introduction to some of the common laboratory techniques including gel electrophoresis.

We went through all the steps from making agarose to micro pipetting a ladder into the wells. We ran a lot of assays and were required to take extensive notes. It was a really great experience, and my favorite laboratory course I’ve taken.

To make the agarose we measured out gelatin powder mixed it with water and microwaved it. This acts as the matrix for which the DNA must travel. The matrix slows down larger sections first, and those bands show up closer to the wells, while the shorter snippets travel further down the agarose matrix. Once the gel is made, it’s placed in a device that electrifies the gels which draws the DNA through the gel (see image above).

Most of the assays we performed required that we test it by running a gel and photographing it and running analysis based on how it fluoresced. This was essential when we were running PCR (polymerase chain reaction) to amplify snippets of DNA to make sure we amplified the section we were seeking.

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