Microbiology Case Study: 64 Year Old Male with Pleuritic Chest Pain and Fevers

Case History

The patient is a 64 year old male with a history of diabetes mellitus and hypertension who presented as a transfer from an outside hospital with a 2 week history of chest pain and pressure, as well as recurrent fevers, rigors, and soaking sweats, and an echocardiogram concerning for a pericardial effusion. He was also found to have markedly elevated CRP, and mildly elevated troponins, was diagnosed with pericarditis, and was started on colchicine. He continued to have fevers, and developed diarrhea and was transferred for elevation of care. C. difficile PCR was negative, and since the onset of diarrhea coincided with the initiation of colchicine, that was determined to be the cause. Blood cultures on arrival grew a Gram positive rod and a transesophageal echocardiogram was done which again showed pericardial thickening with small effusion, and fluid with fibrinous appearance. There was no evidence of valvular vegetation. At this point the patient was started on IV meropenam as he is allergic to penicillin’s and sulfa drugs. The pericarditis seemed to improve with colchicine so a non-infectious process was favored and a repeat ANA was recommended when he has recovered from his current infection.

Laboratory Identification

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Image 1. Gram Stain of blood culture showing gram positive palisading rods.
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Image 2. Gray-white colonies with soft β-hemolysis on blood agar.

Blood cultures grew grey-white colonies that are gram positive, catalase positive rods with soft beta-hemolysis on the blood agar plate, and tumbling motility under light microscopy. CAMP testing would be positive with Staphylococcus aureus. This was identified as Listeria monocytogenes by the MALDI. 

Discussion

Listeria monocytogenes is a gram positive rod that can be found in the soil, water, sewage, vegetation, and as part of the fecal flora of animals. It is facultative intracellular pathogen that is able to invade and survive in human cells including macrophages (1). They possess a surface protein called internalin that interacts with E-cadherin on human cells resulting in endocytosis (1). Once within the cell the bacteria can produce listeriolysin O and other phospholipases which allow it to escape from the phagosome before it fuses with the lysosome, which prevents intracellular killing of the bacteria (2). L. monocytogenes is a common contaminant of food products as it can form biofilms on the food surfaces. Listeria also has the ability to grow a 4°C so it can continue to grow on refrigerated foods (1). Foods such as raw milk, raw vegetables, fish, poultry, and fresh and processed meats are the highest risk for contamination.

Ingestion during pregnancy can result in a flu like illness, occasionally with vaginal discharge, diarrhea, and urinary tract symptoms (1). Infection during pregnancy is particularly dangerous as occult bacteremia with transplacental transmission may occur (2). Infection in utero may result in premature labor and birth of an infected or stillborn fetus. Prognosis is highly dependent on the gestational age at infection.

Non- pregnant adults can also become infected by Listeria. The most common results of ingestion of contaminated food in immunocompetent patients is a transient asymptomatic carrier state, and can be excreted in the feces. Less commonly, febrile gastroenteritis can occur. Immunocompromised patients or those with underlying malignancy tend to present with acute sepsis, meningitis, or meningoencephalitis.  Focal infections such as cutaneous infection, abscesses, arthritis, peritonitis, liver/splenic abscess, cholecystitis, artificial joint/graft infections, osteomyelitis, and myo- and endocarditis can be seen and typically occur in immunocompromised patients as a results of hematogenous spread.1 Treatments includes ampicillin with or without an aminoglycoside. Occasional resistance to tetracyclines has been reported.2

Regarding the patient’s Listeria bacteremia, the patient reported no exposures to the common carriers of Listeria. It is possible that is was translocated from his gut during his diarrhea illness or could have been the cause of his diarrhea, although blood cultures at the outside hospital were negative.

References

  1. Winn, Washington C., et al. Color Atlas and Textbook of Diagnostic Microbiology. Lippincott Williams & Wilkins, 2006.
  2. Tille, Patricia M. Bailey & Scotts Diagnostic Microbiology. 13th ed., Elsevier, 2014.

 

-Casey Rankins, DO, is a 1st year Anatomic and Clinical Pathology resident at the University of Vermont Medical Center.

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-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.

The Best Laid Plans: A “Trial by Fire”

From around 2009 to 2016, I worked very closely with a USA-trained surgeon, Dr. Brian Camazine of Earthwide Surgical Foundation, who visits Nigerian Christian Hospital in Aba, Nigeria for one month every quarter. He performs between 200 to 300 surgeries, which produce 40 to 60 surgical pathology specimens each visit.  Dr. Camazine has invested time, energy, and money into training local Nigerians in surgical skills, acquiring surgical and medical supplies to support his patient population, and following up all of his patients with Skype clinics after he returns.

My role in Dr. Camazine’s activities was to receive the surgical pathology samples, process them, and return results for him as quickly as possible. When Dr. Camazine contacted me, there was no pathology laboratory at NCH. Dr. Camazine uses a heavily subsidized model for all of the services provided at NCH such that a patient may pay ~$200 for a surgery (complete care including pathology) that would have cost them $2,000 to $4000 elsewhere in Nigeria. My hospital at the time had an ongoing project of a similar fashion with several sites in Africa but the costs of that program were growing. Dr. Camazine agreed to pay a fee of $25 per sample to my hospital to offset the technical costs of our laboratory processing the samples, and I provided all diagnostic results pro bono. Dr. Camazine was only charging patients $20 per case for pathology; thus, he subsidized the service further.

I had many long and difficult discussions with Dr. Camazine about this program and how we needed to focus on a sustainable solution that did not involve transport to the US for processing for many reasons including (but not limited to): a) danger and difficulty with sending tissue, b) long turnaround time because of shipping delays, c) chain of custody and requisition challenges, and d) capacity building in pathology. We kept at it with this long-term plan in mind but, as I departed my hospital to join ASCP in 2016, a drastic decision had to be made because I would no longer be able to shepherd this service. Dr. Camazine reached out locally to Nigerian laboratories and was fortunate to meet Dr. Chidi Onwuka from the Department of Histopathology at the University of Uyo Teaching Hospital. Brian and Chidi came to a feasible financial arrangement and, with the closeness of the laboratory, Chidi can return results to Brian in about 1 week (Meet Chidi and read Brian’s Blog here). This was a great success for Brian and Chidi because it represented moving from a non-sustainable, bridging program (i.e., what I had set up with Brian) to a permanent solution with the local laboratory. For over two years, Chidi has provided high quality service with quick turnaround time and massively improved the patient care journey for NCH patients.

On June 27th, 2018, however, that complete pathology solution came to a screeching halt when a fire swept through the laboratory and destroyed all of the equipment and reagents. The laboratory in question had just been completely updated with 40 Million Naira (~$115,000 USD) worth of equipment and upgrades, but it was all lost. Dr. Chidi reached out to Brian, myself, and many others with an urgent request to help him get a replacement laboratory up and running. After so much success, it was heartbreaking to hear such a loss had occurred.

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The ASCP Partners for Cancer Diagnosis and Treatment in Africa Initiative was launched in 2015 with a goal of bringing 100% access to cancer diagnostics services to all patients. Although the population of patients Brian cares for and Chidi diagnoses are within Africa and within the scope of the Partners Initiative, at the time of the fire, there were at least 10 laboratory projects (including equipment, training, IHC, telepathology, etc.) in process through the Partners project. We were seemingly “at capacity” to help. What could we do? Although we have ASCP member volunteers that donate equipment, we have a waiting list of labs wanting to receive the equipment. Although Brian and Chidi are my colleagues and friends, the distribution of global health resources, assistance, and capacity should always be done with equity. As part of the Partners Initiative, ASCP Center for Global Health acquires equipment (typically through donation which means donor requirements of the local countries) and covers shipping costs to move the equipment to the recipient sites but we had not yet formalized this process. But, for Chidi, I simply didn’t have the equipment available to send.

Then, I received a WhatsApp message from Chidi on August 3rd with a small bit of good news. He had located a microtome in the USA that he could purchase; however, he did not have sufficient funds to ship the equipment. Now, finally, ASCP could help him! But it was not quite that easy!

ASCP staff member Dr. Debby Basu got the microtome in the USA to Chidi in Nigeria. This was not an easy task. Debby faced two major challenges for organizing Chidi’s shipment. First, she had to establish key templates and tools necessary to facilitate donation. Although we have several sets of donated equipment that are to be shipped from ASCP to other sites, Chidi’s microtome was the first actual piece of equipment that would go with our new shipping agent. As this was our first shipment with Bollore, she first had to work with Bollore to determine what documentation ASCP was responsible for providing. She then developed the in-house documents, templates and tools needed to facilitate shipment using Bollore’s services (e.g. commercial invoice, packing list, Shipper’s Letter of Instructions (SLI) Form (customs information), donor letters, etc.). She served as the liaison between the original vendor, recipient and shipper to make sure that donation and shipping documentation was consistent, and that information was clear and available to all parties. The second challenge was understanding the complex international shipping guidelines for exporting scientific instruments and goods on US side and importing donation on receiving end. To address this on the domestic side, she worked closely with the shipper directly to clarify domestic customs guidelines specific to the context of the items being shipped and ensure customs documentation was completed appropriately. On the Nigerian side, she connected Chidi to Bollore’s Nigeria-based shipping team to establish a local point of contact for him. She then coordinated with both the US-based and Nigeria-based shipping teams to clarify country-specific importation requirements and provide Chidi with necessary documentation to ensure smooth receipt of instrument. It had been ASCP’s intention to use Bollore for the donation program but Chidi’s emergency pushed our agenda forward and Debby was able to race into action to make the process go. Now, Chidi has his microtome (and is replacing his other equipment) and ASCP’s shipping donation program has its process finalized for the next series of donations.

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ASCP is so grateful to all of our members and member volunteers who have made the Partners Initiative a functional and impactful global health program. We are careful in our assessments, planning, and development of implementation plans with each of our sites and their leadership. However, terrible things happen unexpectantly. We hope that ASCP can always be a light in the dark when all others have gone out.

 

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-Dan Milner, MD, MSc, spent 10 years at Harvard where he taught pathology, microbiology, and infectious disease. He began working in Africa in 1997 as a medical student and has built an international reputation as an expert in cerebral malaria. In his current role as Chief Medical officer of ASCP, he leads all PEPFAR activities as well as the Partners for Cancer Diagnosis and Treatment in Africa Initiative.

A Tale of Two Types

“It was the best of times, it was the worst of times.” In the blood bank, some of the best days can come from some of the worst days. When we come together as a team to work on a puzzling antibody problem, or to respond to a trauma, we can take pride in our work and know we have done our best to help the patient. In the blood bank we are constantly being called upon to learn and to be “disease detectives.” These are the best times. I tell my students that antibody panels are like puzzles and ABO discrepancies are mysteries to solve. Of course, when the Emergency Room is calling for blood for a trauma, or the Operating Room has an emergency surgery on a patient not previously type and crossed, any “problem” to solve can be a bit stressful.

ABO discrepancies are one challenge we face in blood banking. These are generally not clinical problems, but are serologic problems encountered by the blood bank technologists. Some discrepancies are easier to resolve than others, but still usually require a bit of investigation, and time. We don’t see these every day, so they can set us back a step when we do come across them.

One such situation that I recall was a young man in the ER who arrived by ambulance after a motor cycle accident. My trauma beeper went off and I called the ER to see if they wanted blood right away. Typically in these cases we bring them O blood in a cooler, and continue to use type O until we have a blood sample and current type, (performed twice if no prior history) and an antibody screen. In this case we were fortunate in that we got a sample almost immediately, before they started any transfusions. The type and screen was put on our Provue, but the instrument flagged an error on the type. When looking at the gel card, I could see mixed field reactions. Serology results are shown.

Anti-A Anti-B Anti-D Rh cont A cells B cells ABO/Rh
2+mf 0 2+ 0 0 4+ ?

ABO discrepancies occur when unexpected reactions occur in the forward or reverse grouping or the forward typing does not match the reverse typing. In general, RBC and serum grouping reactions are very strong; therefore reactions less than 3+ usually represent the discrepancy. In this case, testing patient cells with anti-A gave a 2+ mixed field reaction and patient cells and anti-D was only a 2+ reaction. The first step was repeating the test with the same sample. The repeat tube typing gave the same results. Additional steps included testing a new sample, completing the antibody screen, which was negative, and reviewing the patient history. At this time, we did have a positive identification on the patient and a medical record number. The patient had no previous Blood Bank history. However, reviewing the ER admission notes, it was noted that the patient had received 2 units of O negative packed cells in the ambulance en route to the hospital. Viewing the anti-A and the anti-D tubes under the microscope confirmed presence of mixed field agglutination.

Mixed field agglutination describes the presence of two populations of red cells. Mixed field agglutination is seen as small or large agglutinates in a field of many unagglutinated cells. In this case, we observed mixed field agglutination with the patient’s own circulating type A positive red blood cells agglutinating with the anti-A antisera, and the type O donor cells he received remaining unagglutinated. Patients can show mixed field reactions after recent out of group transfusions of as few as 1 or 2 units of packed cells. As well, when group O packed RBCs are transfused to a group A, B or AB recipient, there is always a small amount of plasma transfused. Thus, anti-A, anti-B and anti-A,B are almost always passively transferred. Even though it is unlikely that the passively acquired ABO antibodies will cause in vivo hemolysis, it would be recommended to continue transfusing O blood instead of type specific blood for the duration of the immediate episode and until anti-A antibodies are no longer detectable in the patient’s serum.

This case is an example of an artificial chimerism. Chimerism is the presence of 2 cell populations in a single individual and, in this case, was easily explained by the recent out of group transfusions.  This patient was sent to surgery and continued receiving several more units of group O RBCs during and after surgery. The patient’s blood type continued to appear as a mixed cell population during his hospital admission.

There are a number of other scenarios in which mixed field reactions could cause a discrepancy in a patient’s ABO/Rh typing. Some weak subgroups of A (A3) are known for giving mixed field reactions. Mixed field reactions can also be seen in other artificial chimera cases, such as are seen with transplanted bone marrow or peripheral blood stem cells of a different blood type.  If mixed field reactions are present, review the patient’s transfusion history to determine if the patient has been transfused with non-group specific RBC components in the past 3 months or received an ABO-mismatched stem cell or bone marrow transplant. More uncommon and unusual are cases of true chimerism, which can occur with fraternal twins.  Stay tuned for my next transfusion medicine blog for a discussion of chimerism!

A few key tips to remember when encountering an ABO discrepancy:

  • Retest the sample first, using a different method, if available
  • Check for technical or clerical errors
  • Remember that the weakest reactions are usually the ones that are in doubt
  • Complete the antibody screen and note positive reactions
  • Check the patient diagnosis
  • Check Blood Bank history
  • Most of all, take a deep breath and relax. You can solve this!

References

  1. Charles Dickens. A Tale of Two Cities. 1859
  2. George Garratty. Problems Associated With Passively Transfused Blood Group Alloantibodies. AJCP, June 1998
  3. Denise M. Harmening, Modern Blood banking and Transfusion Practices, Sixth edition, 2012.
  4. Christopher Sharpe, et al. Mixed field reactions in ABO and Rh typing chimerism likely resulting from twin haematopoiesis. Blood Transfus. 2014 Oct; 12(4): 608–610.

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-Becky Socha, MS, MLS(ASCP)CM BB CM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 30 years. She’s worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

Innovative Diagnostics for the Developing World

My last two posts have focused on the WHO Essential Diagnostics List (EDL). The EDL, modeled after the Essential Medicines List, is meant to serve as a model guide for countries to use in the development of laboratory services for diagnosis, treatment, and monitoring of common diseases. The EDL is meant to be tailored to an individual nations disease burden, and India is on track to be the first country with a country-specific EDL. I think this is a tremendous step forward for the field of laboratory medicine worldwide, but I do wonder how this will roll out Low or Middle Income Countries (LMIC). How we will got from a list (actually a draft of a list) to real-time diagnostics in the developing world? Let’s take a step back and look at what access to laboratory testing really looks like, and then discuss some innovative diagnostics that might help increase access to useful diagnostics.

In the US, we have many labs tests available – so much so that laboratory utilization has become a very real area of study with groups like Choosing Wisely, PLUGS, and Preventing Overdiagnosis dedicated to helping guide appropriate use of in-vitro diagnostics (IVDs). The US has over 250,000 accredited laboratories, and 18,000 clinical pathologists. That’s about 5.7 pathologists per 100,000 people. In contrast, the continent of Africa has about 1 million people per pathologist, and some African countries have over 5 million people per pathologist. It’s difficult to find a number of laboratories in LMICS, because many are mom and pop type shops that aren’t registered with the Ministry/Department of Health. Test menus are often limited to cell counts and rapid testing, and proficiency testing or quality control is not practiced.

Challenges to building laboratory capacity in LMICs are environment, economics, and education. Environmental challenges include extreme temperatures, limited electricity, and limited access to water. Some point of care options that might be able to work in these environments just aren’t affordable. Many people in LMICS make around $2 USD a day and a $10 iStat/Piccolo/your favorite POC chemistry device cartridge is just out of reach especially if the testing needs to be repeated frequently for monitoring disease progression. Lastly, education remains a challenge as laboratory medicine including not not only technical skills but also the use and interpretation of lab tests is not frequently taught in LMICS.

However, there are many different innovative diagnostics being debuted or in development with these challenges in mind. There are definitely trends in the area of new diagnostics for the developing world. The most distinctive trends are: smartphone imaging, smartphone spectrophotometers, transdermal testing, and paper based sample collection. In my next few posts, I’ll take us through examples of each of these, and I’ll start now with smartphone imaging.

Smartphone imaging is essentially using light boxes, cameras, or apps, to turn a smartphone into an imaging device. Smartphones are even being used as simple ultrasounds! A smartphone microscope can be useful in diagnosing tropical infectious diseases. A good example is the LoaScope, developed by the Fletcher Lab at UC Berkeley. Dr. Daniel Fletcher is known as the “father of the cell phone microscope”. The LoaScope is a mobile phone based microscopy platform plus an app-based algorithm for the detection of L. Loa. The device is elegantly simple: a 3D printed case with a rest of the mobile phone. The case contains a USB port, bluetooth controller board, and LED array, and a carriage for a capillary slide. Blood is introduced to the capillary slide and imaged by the phone. A 5 sec video is captured by the phone, and then analyzed via app that uses a algorithm based on the the wiggling motion of the loa worm. The algorithm actually distinguishes the movement of blood cells as the loa worms move between them! The interpretation of the video lets the user know if the parasite is present or not present. Another bonus the device is that a georeference is captured with every video, which is great for epidemiological studies. In 2017, the LoaScope was used by the NIAID to testing over 16,000 subjects in Cameroon. Because of the LoaScope, over 15,000 subjects were successfully treated with ivermectin without serious complications.

There are some key elements to the LoaScope that are common across the new, innovative diagnostics for the developing world. First, the 3D printed case. 3D printing seems to really be helping this field jump ahead by decreasing the costs associated with creating the physical structure of the devices. Secondly, the device does not require special specimen preparation, nor does it need reagents. Lastly, a simple read-out is a available to the user in real time.

In summary, the EDL is great for telling us what test are, well, essential. Innovative diagnostics are going to help us get to those hard-to-reach places. I look forward to continuing to investigate these with you!

 

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Sarah Riley, PhD, DABCC, is an Assistant Professor of Pediatrics and Pathology and Immunology at Washington University in St. Louis School of Medicine. She is passionate about bringing the lab out of the basement and into the forefront of global health.  

Vending Laboratory Safety

When you put your money into a vending machine, there is always a gamble. There is a risk of the machine not working- it will take your money but not dispense any products, or the item might just get stuck inside the machine and no amount of banging or tipping will help. As humans, though, we take that risk, and the “danger” is only the loss of some money.

The potential danger for a patient in the hospital can be higher. For years, healthcare organizations have been working with other agencies to improve patient safety. Two professions that often serve as the gold standards of safety culture are the airline and nuclear industries. I have seen many speakers over the years from those agencies give amazing speeches on attaining such high safety ratings. On my more cynical days, I often think that hospital caregivers will probably never reach the same level of safety that is seen in the nuclear and airline industries, and I feel there is a “logical” reason for that. If a pilot or an employee at a nuclear plant makes an error, it potentially places his or her own life at risk, so more attention is paid and fewer errors are made. If an employee makes a mistake when treating patients, the error affects the patient and not the employee, so paying constant attention may not seem as urgent to the worker (I told you these were cynical thoughts).

Now let’s go back to the vending machine. There is some risk to take when putting money into the machine, but once the money is accepted, we feel free to make our selection. Now, if you’ve ever watched someone make such a selection, you may notice that they will not risk making a mistake- they will check, double-check, and even triple-check to make sure they press the right button combination so they get the correct item. The outcome of any mistake made here directly affects the person craving that specific soda or candy bar, so the caution taken to ensure a proper selection is greater. Is that just human behavior? Do we make safer choices if the risk directly affects us?

If that theory is true, then laboratory employees should always work safely. They should always wear proper PPE, they should never eat or drink in the labs, and they would never use their cell phones in the department. Yet many lab safety professionals know that these unsafe behaviors still exist, even in today’s world where we handle highly infectious organisms and deal with bloodborne pathogens daily. If unsafe behaviors lead to exposure- to harm that directly affects the employee- why do these behaviors remain? What’s missing from the picture? I believe the answer lies somewhere between complacency and education, but I also believe both can be handled with increased safety awareness.

Staff who have been in the lab for many years can lose their respect for the chemicals and samples they handle every day. They know that they have worked with them for many years with no negative outcomes, and older lab employees remember the days when all of those unsafe behaviors ran rampantly. Ask a mature lab tech about smoking in the lab, placing party casseroles in the microbiology incubator to keep it warm for the party, and even mouth pipetting. Many laboratory employees worked in environments like that and came out unscathed. But not everyone did.

The reason OSHA and other lab accrediting agencies put forth more stringent safety regulations over the years is because so many lab employees were infected, injured, or killed as a direct result of those unsafe actions. Even in the span of my ten years in lab safety, I can tell a different horror story to each person who says they are fine not paying attention to safety rules. It’s important to do that. Injuries and exposures occur every day in labs, and if they happen in your lab, it is vital the story is told to other staff. Transparency and discussing methods of prevention with staff makes an impact because it makes the danger real and more personal. If you’re in a lab where accidents are rare, that’s great- but make sure you continually raise awareness of the inherent dangers in the lab work place by finding stories of events in other labs and talking about them. Tell stories of near miss events as well. It is good to discuss events that were averted through solid safety practices as well.

Lab safety education, both initial and on-going, are key to helping staff understand the environments in which they work. Safety competencies, drills, and tests are good tools to keep awareness of the lab’s safety issues on the minds of employees every day. Telling safety stories and sharing incidents are other actions that can also reduce safety complacency. Every day our employees come to work, and the potential dangerous possibilities are always there in the lab “vending machine.” Help them to be careful to make the correct selection so they can remain healthy and happy with the career choice they have made.

 

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Dan Scungio, MT(ASCP), SLS, CQA (ASQ) has over 25 years experience as a certified medical technologist. Today he is the Laboratory Safety Officer for Sentara Healthcare, a system of seven hospitals and over 20 laboratories and draw sites in the Tidewater area of Virginia. He is also known as Dan the Lab Safety Man, a lab safety consultant, educator, and trainer.

Laboratory Medicine for Transgender Patients: An Introduction

Welcome to a new series where I’ll explore the role of lab medicine in the care of transgender patients! Many of you may be asking yourself, “Why should I care? I’m in the lab far separated from these dicey patient care issues.” However, the lab plays important roles as the patient moves through the healthcare setting. Everywhere from name confirmation by phlebotomists and before blood transfusion to sex-specific reference intervals, the lab interacts with the healthcare of transgender patients in important ways. With more transgender patients presenting for clinical management, and more clinicians armed with hormone therapy guidelines created and endorsed by the Endocrine Society, it will be expected for laboratory professionals to know how to manage these patients too.

For me, my first encounter with transgender healthcare through the laboratory was during my clinical chemistry rotation when the lab paged me about a very high estradiol value 10 times higher than the upper limit of normal. I found that the patient was a transgender woman taking excessive hormone doses. Their doctor counseled them and persuaded them to stick with their prescribed dose, because the risks of supraphysiologic estrogen is not known. While we were glad the patient didn’t have an estrogen secreting tumor, I wondered how this hormone therapy may affect other aspects of their health and physiology as reflected by lab values.

After a literature review, I found there were few studies that addressed changes in lab values with hormone therapy. Those papers I found had limited numbers of patients, so I decided to find the answers for myself. Subsequently, I (along with two medical students) studied a large number of patients attending transgender specific clinics.  I’ll discuss our findings as a part of this series.

For now, I’ll go over terminology so everyone can be on the same page. Many of us are likely unfamiliar with the experiences of transgender individuals and don’t realize how what appears to be a verbal misstep can be offensive. The first distinction to make is the difference between sex assigned at birth and gender. Sex is assigned at birth to a child, often based on external anatomy. Gender is the set of behaviors and roles that society or culture assigns to a person that ranges from masculine to feminine. However, gender identity is a deeply held internal sense of whether you consider yourself male, female, both or neither. This is distinct from sexual orientation, which one colleague explains: “orientation is who you go to bed with, gender expression is what you go to bed wearing, and gender is who you go to bed as.” When one’s gender identity is concordant with their sex assigned at birth, they are called cisgender; whereas, discordance between sex assigned at birth and gender identity is termed transgender (I think of cis and trans stereochemistry in organic chemistry). The process of using medical or surgical interventions to transition is referred to as gender-affirming hormone therapy or gender-affirming surgery.

The easiest way to address someone whose preferred name doesn’t match their sex in their record is to address them as they appear: use female pronouns if they are dressed as a woman and male pronouns if they are dressed as a man. And if you’re not comfortable with that, a simple “How would you like to be addressed?” is appreciated. I will go into the importance and challenges of legal sex/name and pronouns in the electronic health record in a later discussion.

To round out the topic of terminology, I’d also like to mention a few terms that should be avoided. “Transgendered” adds an unnecessary “-ed” as transgender is already an adjective. It is further confusing, because it makes the word sound past tense (we wouldn’t say “lesbianed,” for example). Rather, a person undergoes gender transition as they accept and express their gender identity through a set of social, physical, medical or legal changes (sometimes call gender affirmation process). Using terms like pre-op/ post-op/ sex change overly emphasizes the role of surgery in the process, and thus gender transition is more inclusive. Similarly, asking for someone’s “real name” overly emphasizes their legal name and there are limited situations where that would be necessary to use. Derogatory terms include tranny, hermaphrodite, or transvestite and shouldn’t be used even when referring to people who are intersex or wear clothes of the opposite sex.

Thanks for making it all the way through this first post, I look forward to hearing any questions you have and exploring this topic together further!

References

  1. Goldstein Z, Corneil TA, Greene DN. When Gender Identity Doesn’t Equal Sex Recorded at Birth: The Role of the Laboratory in Providing Effective Healthcare to the Transgender Community. Clinical Chemistry 2017; 63(8):1342-1352.
  2. Rosendale N, Goldman S, Ortiz GM et al. Acute Clinical Care of Transgender Patients. JAMA Intern Med. Published online August 27, 2018.
  3. Roberts TK, Kraft CS, French D et al. Interpreting laboratory results in transgender patients on hormone therapy. Am J Med. 2014;127(2):159-62.

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-Jeff SoRelle, MD is a Molecular Genetic Pathology fellow at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and advancing quality in molecular diagnostics.

Data Analysis for NGS by Ion Torrent – Part One – How Did the Run Perform?

Here comes the fun part.  It’s taken a day for library prep, an overnight run for the clonal amplification; the next day includes loading the chip with the ISPs and then running the chip on the sequencer.  After the chip has run on the sequencer, the data is pushed from the sequencer (the PGM) to the server connected to the sequencer.  This aspect of NGS surprised me – the size of the files is amazing – for one 316 chip, the file that includes all of the raw data averages about 100GB.  To deal with this amount of data, the server attached to the sequencer is 12TB, and even still we have to have a procedure to deal with removing files off that sequencer to keep space for future runs.

Anyway, the raw data is pushed to the server and the data analysis begins.  The Torrent Suite Software first analyzes the ISP info, as shown in the graphic below.  It gives a “heat map” of the chip (the football shape) in which red means the wells in those areas were full with ISPs.  Yellow means there are fewer ISPs and blue means there are none.  So, you can see below, there is a small area of blue within the football shape – this area did not have any ISPs in it.  92% of the wells on this chip were filled, however, which is about the max a chip can be loaded.

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These ISPs are then broken down into categories.  First, how many of the wells had ISPs in them – here, 92.5% of the 6,337,389 wells contained ISPs.  Of those ISPs, 99.8% of them have product on them that can be sequenced (Live ISPs).  Of those Live ISPs, 0.4% of them contain control Test Fragments and 99.6% of them contain actual patient sample library amplicons.  The Test Fragments are spiked in prior to sequencing and act as a control to evaluate how the sequencing run performed.  Lastly, the ISPs that contain patient sample library amplicons are analyzed.  Those ISPs that contain more than one amplicon (say it has an amplicon of EGFR Exon 19 and another specimen’s amplicon of KRAS Exon 2) give mixed signals and cannot be analyzed, so they are thrown out of the data analysis and into a bin called “polyclonal”.  Low quality ISPs are also thrown out – anything that did not pass the thresholds for quality.  And lastly, ISPs that only contain adapter dimers are thrown out.  For a run of AmpliSeq Cancer Hotspot Panel v2 specimens, most of which come from FFPE specimens that are low quality to start with, a run that contains over 50% Final Library ISPs is actually a very good run, interestingly enough.  The 316v2 chips are rated to sequence 1 million reads (each ISP yields one read), and on this example run, over 3 million reads were sequenced, so this is a successful run.

After the ISPs are analyzed and the high quality ones are kept, the analysis goes on.  The Torrent Suite software then calls the bases based on the raw flow data.  These bases are then aligned to a reference, in our case hg19, a commonly used human genome reference.  Quality scores are assigned at this point.  A Phred-based quality score is used for NGS, shown in the table below.

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Lastly, the reads are put into bins based on the barcode that was used for each patient specimen – remember the small part of the adapter that was added in library prep so that the specimens could be mixed together?  The software reads that adapter sequence then assigns each read based on those sequences.  The specimens should all have approximately the same number of reads since they were normalized to the same concentration at the end of library prep, but there may be some variability due to specimen quality, as you can see below.

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In next quarter’s post, we will dive into the individual specimen results!

 

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-Sharleen Rapp, BS, MB (ASCP)CM is a Molecular Diagnostics Coordinator in the Molecular Diagnostics Laboratory at Nebraska Medicine.