Category: generalist

Next Generation Sequencing and Personalized Genomic Patient Care

I’m sure that everyone has heard about next generation sequencing (NGS). But why exactly is it a big deal? Even though I have spent a significant amount of time at the bench performing “wet lab” basic science research and was acquainted with the term, I did not have practical hands-on experience with NGS prior to residency. It was not a readily accessible technology during my biomedical research days prior to medical school and so I did not entirely grasp the full power of this then disruptive technology until I was a pathology resident, and even more so, as an applicant for molecular genetic pathology (MGP) fellowships.

All of us should have previously learned about the “gold standard,” Sanger sequencing. This method combined irreversible dideoxynucleotide chain termination with a detection method such as gel electrophoresis, or on a larger, automated level, capillary electrophoresis. It was powerful because it allowed us to read the genomic map that directs cellular life, albeit only one sequence at a time. It served as the mainstay for more than a quarter of a century and still is employed for smaller scale sequencing or for long (>500 bp) stretches of DNA.

In the 1990’s, initial methods of massively parallel signature sequencing (MPSS) and pyrosequencing began to appear which would lay the groundwork for today’s massively parallel sequencing (MPS), also known as second generation sequencing, or more popularly as NGS. The two most commonly utilized NGS platforms to date are based on semi-conductor technology for the Ion Torrent (now Life Technologies) and reversible dye-terminator, sequencing based synthesis (SBS) technology for the Illumina platforms.

The power of NGS comes from its ability to simultaneously sequence 1 million to 43 billion short reads (400-500 bp each). The Human Genome Project took over a decade to complete and cost nearly $3 billion whereas NGS can sequence the same genome for a cost on the order of $1000’s, a cost that is further decreasing as the technology is refined. When I was working in research (eons ago), we had nitrocellulose based dot arrays where each “dot” represented multiple copies of a specific cDNA sequence and which helped us to build expression profiles for our particular area of study. This would be analogous to analog technology and NGS would now be considered a digital version.

With conventional PCR, we could only amplify one target sequence per sample reaction. The results were also only qualitative. The results only measurable after multiple cycles of denaturation, annealing, and extension as amplified product of the expected size or no amplified product. Then came along real-time or quantitative PCR (qPCR) which some people refer to also as RT-PCR (although this is a confusing term for people like me who were around before qPCR and think of RT-PCR as meaning reverse transcription PCR). The power of qPCR was that within the linear range of detection, we could now quantitate the amount of product present in real time. NGS also provides the resolution of qPCR in terms of quantitation.

So, as a technology, NGS nicely combines and refines some (but not all) characteristics of multiple technologies with large scale profiling. But for a non-molecular person, what is the relevance? Obviously, it has taken us some time to get to this point, even though the Human Genome Project was completed in 2003 (it started in 1990). We needed time for biomedical and translational research to provide us with clinical significance to the mutations and genetic aberrations NGS could identify. We also needed to develop tools to distinguish true mutations with clinical significance from benign polymorphisms present in our population and to build our bioinformatics support.

But here we are, stepping into the new frontier of personalized genomic medicine. Sure, there is a lot of hype surrounding it and these promises will take time to keep. But these are exciting times for someone like me who fell in love with the molecular dance that plays out within our cells. One of the reasons that I want to complete an MGP fellowship is to get more hands-on practical knowledge of the nuances of NGS from a technical standpoint but also to collaborate with other physicians in directing patients to the correct clinical trials and targeted treatment – and therein, is where the power of NGS really lies. For someone who may never get to meet the patient, at least for me, there’s probably no greater satisfaction than knowing you had a pivotal part in helping a patient more effectively combat their disease. Personalized genomic medicine is another step in that direction.

[12/12/2014: edited to fix a few inaccuracies. We apologize for the error.]

Chung

-Betty Chung, DO, MPH, MA is a third year resident physician at Rutgers – Robert Wood Johnson University Hospital in New Brunswick, NJ.

Lab Myths: Do You Know Why You Do Things?

Do you ever wonder how many of the things we commonly do in the lab are based on verifiable, objective data? A good way to discover the practices that I call lab myths is to teach laboratory medicine. Any student worth his or her salt will ask you why you perform tasks a certain way, and occasionally you will be unable to locate a good reason. Examples of this type of common practice abound and the following paragraphs mention a few.

Why do you wipe away the first drop of blood from a finger or heelstick for most uses? Everyone knows wiping away the first drop is the correct way to do a capillary collection, because the first drop of blood is contaminated with disinfectant and/or tissue fluid or tissue, etc. That only makes sense. Okay. My question for you is this: where’s the data that proves that? Has anyone ever actually analyzed the glucose, or sodium, or potassium, in the first drop, then the second drop, then the third drop and shown that there is a statistically different result, enough that you should not use the first drop? Or is it a lab myth? We do it because it makes sense and we’ve done it for as long as anyone can remember and everyone does it, but I have been unable to locate any actual data to support the practice. (And if you ask a group of diabetics, most of them use the first drop!)

Here’s another example: Blood gas samples should be sent to the lab on ice to prevent sample degradation. This is another common lab practice that everyone “knows,” and with this one you can find supporting data in places. Yet there is also data to suggest that if the sample is to be analyzed within 30 minutes, ice is unnecessary. In addition, many of those early studies were done on blood gas samples collected in glass syringes. There is data to suggest that there are different dynamics in samples collected in plastic syringes (1). When was the last time you received a glass syringe in your laboratory? The last glass syringe I’ve seen was filled with Novocaine and coming at me at the dentist office.

A third lab myth: 60 – 70% of all medical decisions are based on laboratory test results (2). You can find this statistic quoted everywhere in laboratory medicine (Mayo, CAP, Modernising Pathology Services document on UK Department of Health website, IFCC, to name a few). I’ve always wondered where the data is that supports this assertion. Granted I agree that it makes intuitive sense that the majority of medical decisions are based on laboratory data, however, has anyone actually demonstrated it with supporting data? It also makes sense that 95% of all statistics are made up on the spot.

These lab myths are examples that illustrate a point. Sometimes what we “know” can cause us not to question things we should question, and investigations are not performed that should be performed. It’s a good idea to always question a long standing practice because, like in the blood gas example, conditions may have changed (Who still uses glass syringes?). So think about it. Do you have your own lab myths?

  1. Mahoney JJ, Harvey JA, Wong RJ, Van Kessel AL. Changes in oxygen measurement when whole blood is stored in iced plastic or glass syringes. Clin Chem 37(7):1244-48. 1991.
  2. Forsman RW. Why is laboratory an afterthought for managed care organizations? Clin Chem 42(5):813-16. 1996.

 

-Patti Jones PhD, DABCC, FACB, is the Clinical Director of the Chemistry and Metabolic Disease Laboratories at Children’s Medical Center in Dallas, TX and a Professor of Pathology at University of Texas Southwestern Medical Center in Dallas.

Pediatric Labs are Different

Why do I say pediatric labs are different? A blood test is a blood test, right? Of course it is, however there are a lot of aspects of operating a pediatric lab that really are different from the way operations run in a lab whose clientele is mainly adults.

Most people can think of the most obvious difference: age-related reference intervals. The concentration of various biomarkers in the body changes as an infant grows and develops into an adult. In some cases, there’s not a lot of change in the biomarker. A blood pH is pretty constant over the course of a person’s life, as are the electrolytes. The reference intervals for these may be broader in infancy, but in general they don’t change a lot. However some biomarkers change so drastically that normal levels in childhood would be considered pathological in an adult. Without reference intervals tied to age or developmental state, these tests are not able to be properly interpreted. Alkaline phosphatase during bone growth and steroid hormones during puberty are two good examples of this.

The things that people tend not to think about that cause a pediatric lab to be different are predominately all centered on issues with sample volume. Especially in infancy, children have limited blood volume for testing, and this fact affects nearly every operation in a pediatric laboratory.

Front end processing of samples is affected, as often more than 60% of the tubes arriving in the lab are microtubes, which hold 1 mL or less. These tubes may not be able to hold a barcode label, nor fit on an instrument robotic system. The sample must be aliquotted into tubes that will fit a system, or hand fed into an instrument, and often hand-programmed into the instrument. More manual steps results in more opportunities for human error. In addition, the amount of sample used by any given instrument for testing must be considered. That sample volume includes the actual volume necessary for analysis, plus the volume necessary to run HIL (hemolysis, icterus, and lipemia) indices and the instrument dead volume (the volume below which an instrument cannot pipet the sample). Instruments with large dead volumes will not be found in pediatric labs.

Small sample volumes also affect the ability of the lab to add-on tests to samples already in the lab, or rerun samples later to check results. There may be insufficient volume to run more tests or rerun tests, or the sample may have evaporated and be unacceptable for running additional analyses. A study done using a 5 mL serum sample and a 0.1 mL serum sample and allowing both to sit open to the air for 4 hours showed that the 5 mL serum sample had less than 10% difference between original test values and those run 4 hours later. The 0.1 mL sample had more than 50% difference in its values. In addition, with just enough sample to run a test one time, if a dilution needs to be made, it may not be possible.

Lastly, there are a few test menu differences in a pediatric laboratory. Tests commonly found in pediatric labs, but essentially never in predominately adult labs include things like testing for inborn errors of metabolism and sweat chloride testing for Cystic Fibrosis. Conversely, common tests in an adult lab that are not performed in a pediatric lab include serum protein electrophoresis, PSA and other tumor markers. Some tests are performed in both labs but for different indications. For example, AFP is used as a tumor marker for hepatocellular carcinoma in pediatrics instead of as a maternal prenatal screening tool like in an adult lab.

These are some of the ways in which pediatric lab medicine differs from adult lab medicine, and offers unique challenges in day to day operations.

 

-Patti Jones PhD, DABCC, FACB, is the Clinical Director of the Chemistry and Metabolic Disease Laboratories at Children’s Medical Center in Dallas, TX and a Professor of Pathology at University of Texas Southwestern Medical Center in Dallas.

ASCP 2015 call for Proposals

ASCP 2015 ANNUAL MEETING – CALL FOR PROPOSALS
The 2015 Call for Proposals is now open! The ASCP 2015 Annual Meeting will be held in Long Beach, CA October 28-30. If you submitted a proposal for the 2013 or 2014 Annual Meeting, you can log in with your same username and password. You can edit and resubmit your previous proposals and add new ones. The deadline date is Monday, December 8th.

Please inform any co-presenters that you will be submitting a proposal as they will receive an email asking for demographic and disclosure information right after you submit the proposal.  Complete the proposal at ASCP 2015.

Educating the Doctors

If you had a chance to spend one day with a group of fourth year medical students who had already been accepted into residency programs, and you had the goal of providing them with the information any beginning doctor needs to know about the laboratory, where would you begin and what would you teach them?

I had this opportunity recently. The director of a medical school boot camp for Fourth-year medical students (MS4) who would start residency in two months approached me, wanting to know if I’d like this opportunity. Of course, I jumped at the chance. The hardest thing was deciding what information to leave out, to essentially focus the short course on the minimum information related to the lab that a doctor should know when they begin their career. I can honestly say that the opportunity was educational for me also – it showed me exactly how little a graduating doctor knows about the lab! Now in its third iteration, we learn and add and subtract as we go.

We do a brief introduction and overview of general lab structure and then start with phlebotomy. Most doctors (and I’m going to exclude everyone who entered medical school after being a medical technologist) have no idea that the tube top color indicates the type of anticoagulant, and for instance that every purple top tube everywhere in the world has EDTA anticoagulant in it.  We also covered basic phlebotomy technique. Then we rotated them in groups through the various sections of the lab, allowing each section to educate the group on the some of the items they considered the most important features of that section. Some of the topics that were covered include:

Client services/accessioning: some tests utilize a whole blood sample (CBC, blood gases), many, many samples require spinning and aliquotting while maintaining sample identity. Hemolyis, lipemia and icterus interfere with tests.

Chemistry: batch vs random access testing, main chemistry analyzer vs manual testing, pre-analytical affects on test results; reference intervals

Hematology: why clotted tubes can’t be used, how white cell differentials are performed (mostly manual in pediatric institutions)

Microbiology: blood culture bottles in the instrument vs plating and identification; how susceptibility testing works; likelihood of a false positive on a positive flu test run in the summer

Blood banking: what a type and crossmatch includes; how various blood products should be transported; uncrossmatched blood availability

Each section is also instructed to encourage questions and interaction with the MS4s as they tick off main points.

This is an educational opportunity I wish I were granted for all MS4s everywhere. Each year we run this program we refine it as we learn what they most need to know, as well as what they don’t know and what we don’t know. It’s a wonderful learning process.

 

???????????????????????????????????????????????????????????????????????????????????

-Patti Jones PhD, DABCC, FACB, is the Clinical Director of the Chemistry and Metabolic Disease Laboratories at Children’s Medical Center in Dallas, TX and a Professor of Pathology at University of Texas Southwestern Medical Center in Dallas.

 

 

The Unsung Heroes

I have been very pleased to see our professional societies, such as ASCP, become truly active and engaged in bringing attention to the field of pathology, reminding our clinical colleagues that we are in no way the “Doctors-of-the-Lesser-God.” We certainly represent a valuable part of the healthcare team even if our care is provided in a more indirect than direct fashion.

Indeed, I applaud this effort, however, there seems to me to be another missing element that we pathologists, not just our professional society, should embrace. I would hope that we look to expand this to acknowledge the significant role our laboratory staff plays each day on behalf of patient care. The laboratory staff, whether certified MTs, MLTs, phlebotomists, or administrative personnel are the unsung heroes, often forgotten or neglected and without recognition for their much-needed skills and responsibilities. Our laboratory staff represents the legs upon which we stand.

Sadly enough, in my many years in private practice and subsequent consulting, it is apparent to me that pathologists often have very limited interaction with the staff outside of the Histology/Surgical Pathology suite. This is unfortunate as it limits us both professionally and personally. Some of my favorite memories and shining moments from my practice were those that involved getting to know and being a part of the lab team. There is nothing more rewarding than feeling you have learned and participated alongside these co-workers! And there is nothing sadder to me than hearing laboratory staff members say that they have not laid eyes upon a pathologist in weeks or see their physicians only if they seek them out.

Pathologists should be actively interacting with staff in all areas of the laboratory, whether Surgical or Clinical, fostering good relationships and also acting as ambassadors for these staff and their services. We should encourage our clinical colleagues to understand the importance of this group and utilize their expertise as part of the medical team. This helps us all to grow and learn via sometimes differing perspectives which work together to bring quality patient care.

So, while we are utilizing our professional society to grow our own outreach and highlight the important role of pathologists, let us not forget to include our laboratory staff members and what they bring to the table. Make every day the day to support one another and put our cumulative best efforts to quality safe laboratory practice and patient care.

Burns

-Dr. Burns was a private practice pathologist, and Medical Director for the Jewish Hospital Healthcare System in Louisville, KY. for 20 years. She has practiced both surgical and clinical pathology and has been an Assistant Clinical Professor at the University of Louisville. She is currently available for consulting in Patient Blood Management and Transfusion Medicine. You can reach her at cburnspbm@gmail.com.

Podcast: Answers to Your Ebola Preparedness Questions

The editors of Lab Medicine recently sat down with ASCP President Dr. Finn, Dr. Nancy Cornish from the Centers for Disease Control, and Dr. Lance Peterson from NorthShore HealthSystem to answer your questions about laboratory preparedness for a patient infected with Ebola Virus. You can listen to it here.