A Trilogy of Food, Fun, and Facts: Musings of a Pathologist-in-Training

Pathology is a perfectly blended specialty filled with food, fun and a whole range of factual morphological descriptions!

As a pathology resident, one of the first things that got me intrigued by the specialty was its strong association with many food epithets. From the almond-shaped ovary,1 to the blueberry muffin baby,2 to the coffee bean nuclei in the thyroid,3 fried egg appearance of mast cells,4 grape-like lesions seen in molar pregnancy5 to the flat cake placenta6 and even to the strawberry cervix!7 The list is endless. I found these descriptions so interesting that I kept asking myself, “why do pathologists have to make associations with food for many normal and pathological disease processes we see around?”

Aside from the fun association with food (which I happen to like a lot), getting to learn and understand the pathology of disease processes, genetic and syndromic associations have been a fascinating, humbling, and altogether nerve-wracking experience for me.

It has been fascinating because I totally enjoy learning about the underlying processes that get some people sick while others stay healthy. At the same time, it has also been humbling, because, then I realize that so many disease processes are genetically determined and so out of our control. Along the same lines, the experience has also been neck-wracking, because of the detail and efficiency that goes into mastering different disease morphologies and preparing a comprehensive pathology report. The ability to tell the difference between two very similar disease entities but with different morphological features can drive one crazy, because, sometimes everything just seems to look the same!

I remember my early days as a resident. The first week in residency training to be precise. Then, I got reintroduced to the microscope, which is the power of the pathologist. Looking into the microscope and feigning to see what the senior residents and attendings were seeing felt like outright torture to me. You know why? It’s because everything under the microscope was either blue or pink.

In my few years of training as a resident, I have come to learn that in order to be successful as a pathologist, one must be adept with every single detail. As Pathologists, we deal with the facts. We do not make things up, and strive to present the facts of every case which ultimately supports our rendered diagnoses.

Unlike when I first started my residency training, I now know that not everything under the microscope is just blue and pink, and even if they are indeed blue and pink, the degree of their “blueness” or “pinkness” varies. And the intensity of the hematoxylin and eosin (H&E)/immunohistochemical stains may sometimes tell disease entities apart from one another. So, sometimes when people ask me what type of doctor I am training to be, I tell them, “I am a doctor of colors,” which of course often leaves them confused!

I also tell people that I am training to be a doctor who works from behind the scenes, to make sure they get treated right all the time. And this realization I believe is what has created the greatest impression for me. Realizing that a patient’s choice of treatment may totally be dependent on the pronouncements I make on their disease process, is something that gets me motivated to keep putting in my best into my training in order to become one of the best in my field. Therefore, even though we operate as doctors from behind the scenes, our professional judgments often go a long way in impacting the welfare and outcomes of patients whom we never get to see, which is one of the aspects of the specialty that I truly love.

So, pathology as a specialty has given me a more robust meaning to life. I have learned to value and appreciate the time I spend with those I love, and to make special moments with them count. It has made me realize that there are certain things about life such as genetic diseases, that I have no control over and therefore should only be concerned with giving my very best all the time. Pathology has also made me more detail oriented, by learning to distinguish benign from malignant processes. It has reinforced for me, the importance of being the best person I can be to both my family, neighbors and my community in general. And I would also add that pathology has further reignited my love for good food. So, let the party begin!!!

References

  1. Ignatavicius DD, Workman ML. Medical-Surgical Nursing: Patient-centered collaborative care. Elsevier Health Sciences; 2015. 1735p.
  2. Mehta V, Balachandran C, Lonikar V. Blueberry muffin baby: a pictoral differential diagnosis. Dermatol Online J. 2008;14(2):8.
  3. Oertli D, Udelsman R. Surgery of the thyroid and parathyroid Glands. Springer Science & Business Media; 2012. p. 620.
  4. Bolognia JL, Jorizzo JL, Rapini RP. Dermatology. Gulf Professional Publishing; p. 1438.
  5. Daftary. 100+Clinical Cases In Obstetrics. Elsevier India; 2006. p. 478.
  6. Power ML, Schulkin J. The Evolution of the Human Placenta. JHU Press; 2012. p. 278.
  7. Swygard H, Seña AC, Hobbs MM, Cohen MS. Trichomoniasis: clinical manifestations, diagnosis and management. Sex Transm Infect. 2004 Apr 1;80(2):91–5.

-Evi Abada, MD, MS is a Resident Physician in anatomic and clinical pathology at the Wayne State University School of Medicine/Detroit Medical Center in Michigan. She earned her Masters of Science in International Health Policy and Management from Brandeis University in Massachusetts, and is a global health advocate. Dr. Abada has been appointed to serve on the ASCP’s Resident’s Council and was named one of ASCP’S 40 under Forty honorees for the year 2020. You can follow her on twitter @EviAbadaMD.

Patients and Patience (Part 2)

Holiday season in well behind us and, while we celebrate and coordinate getting our COVID vaccinations (side note: get yours please), I’d like to revisit a piece from a while back called “Patients and Patience.”

Then I talked about how our professionally shared spirit of camaraderie and patient advocacy go hand-in-hand with the ASCP mission. How, regardless of what role we play in patient care, we continue to give as much as we can to make the lives and hopes of patients everywhere a bit brighter. This year especially, as the pandemic continues to take over all frequencies and channels (including this blog, I’m sorry), I think it’s especially poignant to remember how life can be both grand and fragile. You’ve read my musings on how doctors can be patients too, and how we can all be stretch so thin it can affect our health. When you think the experiences of anyone in healthcare this past year you can’t help but reflect on how burnout and compassion really both know no bounds.

This month, I’m dedicating this piece to one of Loyola’s faculty who sadly and unexpectedly passed away just before the New Year, Dr. Stefan Pambuccian. In a caustic reminder of life’s grand fragility, he was an archetype of what it meant to be an accomplished and respected pathologist, physician, teacher, and friend. While he and other faculty here push all of us resident/trainees or fellows to be better, and research, publish, learn, share, and grow, people like Dr. Pambuccian set the tone with years of experience, an open door, and an uncanny ability to give you a differential diagnosis from only peeking at a slide from across the room at 1x—not a typo.

Image 1. You can barely find any posters on our walls (of which there are many) that don’t in some way bear the name or relate to the work or Dr. Pambuccian here at Loyola. His office and door continue to receive new messages of loss, praise, thanks, and sentiment. Losses like these are never easy, but no one here went through this alone.

While the residents were having a great uplifting secret santa exchange a few days after Christmas, we went around praising our anonymous gift recipients and shared some laughs amidst a new warm holiday memory. The same joy that filled our workroom vanished after everyone had heard the awful news, taking time to process and simply be with each other that afternoon and the weeks that followed. That’s exactly the message I think rings true this time around: in order to care for patients, and ourselves, our friends, and our colleagues, we should always have reserves of patience, compassion, and humanity. While there are great wellness programs, and tips and tricks to avoid burnout, that’s for other blogs; sometimes what one really needs is other people. Peers. Friends. Family. I’m relatively new here in this program, but what I could see that day was an immediate working shift from signing out cases to taking the time to make sure everyone was okay—whatever that meant for them. You can promote wellness all day, but you can’t (ethically) pose any actual testing of resilience. The loss of Dr. Pambuccian not only demonstrated the camaraderie and compassion at Loyola Pathology, but made sure we all learned what it means to be a great pathologist.

Image 2. A fun yet fleeting secret Santa. Despite the mood of this day being changed by the news, happy memories are still just as important. Both the sad and happy parts of this day brought us all closer, and stronger, together.

Like I said, my interactions with him were brief at best but he gave the morning didactic at my very first residency interview here and I learned all about his bottomless sense of humor and wit. Since starting, he was always there running the Thursday unknown sessions, where I felt empowered to participate alongside his openness for learners at all levels. I even remember I was on-call one night with him on service, and after checking in with other residents, I gave him a call to say there was nothing much happening tonight—I barely made it past my hello, before he told me to have  good night because he already checked the surgery schedule and was just waiting on me to call. Thanks. I could never do justice in telling stories about him when compared to literally anyone else in my department. There were countless more stories, and tons of experiences my fellow senior residents and faculty all shared about their working with him. I just feel lucky enough to have known him.

Image 3. I volunteered along with one of our fellows to take new faculty and resident photographs for our new website. My cloud photo storage is full of 3-4 similarly posed faces of everyone I work with…except Dr. Pambuccian. He wanted to make this fun, much like everything else he did.

I find myself in the same position as the last time I talked on this topic: at a new chapter in life to start becoming the doctor I set off on this journey to become many years ago. With the addition of excellent faculty mentors, friends and colleagues, and an ongoing, renewed sense of purpose, I’ll keep you all posted.

To read more about Dr. Pambuccian’s life, his love of art and cats, his numerous publications which will undoubtedly crash your computer, please click this link to Loyola Pathology’s in memoriam.

Thank you for reading and letting me take this aside to say, as I have before, that we deserve the same compassion and patience as we extend to our patients and that the values that inspire us to do our best to improve healthcare at large are the same values that can help us build strong, caring relationships with our families, friends, and colleagues.

Take care of yourselves and those around you. Thanks for reading! See you next time!

(And look into how and where to get your COVID vaccine!)

Constantine E. Kanakis MD, MSc, MLS(ASCP)CM is a first-year resident physician in the Pathology and Laboratory Medicine Department at Loyola University Medical Center in Chicago with interests in hematopathology, transfusion medicine, bioethics, public health, and graphic medicine. He is a certified CAP inspector, holds an ASCP LMU certificate, and xxx. He was named on the 2017 ASCP Forty Under 40 list, The Pathologist magazine’s 2020 Power List and serves on ASCP’s Commission for Continuing Professional Development, Social Media Committee, and Patient Champions Advisory Board. He was featured in several online forums during the peak of the COVID pandemic discussing laboratory-related testing considerations, delivered a TEDx talk called “Unrecognizable Medicine,” and sits on the Auxiliary Board of the American Red Cross in Illinois. Dr. Kanakis is active on social media; follow him at @CEKanakisMD.

Case Study: Newborn with Thrombocytopenia and Bruising

A newborn, healthy, full term, male child, was born with bruising on his left thigh and developed petechiae and purpuric hemorrhages several hours after birth. The baby was moved to the NICU for observation and a CBC was ordered by the NICU provider.

  • WBC, RBC, Hgb, Hct and indicies were normal
  • Platelet count 58 x103/μL
  • Baby exhibited no symptoms of sepsis
  • Smear reviewed with no platelet clumping observed

The mother is a 28 year old, gravida 1, para 1 with normal CBC and platelet count. Her prenatal history was unremarkable. She has no history of immune thrombocytopenia (ITP) and no history of being prescribed drugs known to be associated with drug induced thrombocytopenia

Thrombocytopenia is not an uncommon finding in neonates, particularly in the neonatal intensive care unit (NICU). In preterm infants, the most common causes of thrombocytopenia are complications of pregnancy, including pregnancy-induced hypertension (PIH), intrauterine growth retardation, preeclampsia ,and HELLP syndrome (hemolytic anemia, elevated liver enzymes, low platelet count). Examination of a peripheral smear in these patients will typically reveal neutropenia with densely packed red cells, increased nucleated RBCs and deceased platelet estimate. These placental insufficiency cases typically occur within the first 72 hours of life, platelet counts are >50 x 103/μL, resolve without treatment and require no further investigation. On the other hand, thrombocytopenia in preterm infants that develops after 72 hours is most likely due to sepsis or necrotizing enterocolitis and requires investigation and treatment.2

In an otherwise healthy appearing full term infant, the most common cause of thrombocytopenia in the first 72 hours of life is neonatal alloimmune thrombocytopenia (NAIT). When a platelet count drops below 150 x 103/L in these newborns, it is important to investigate the thrombocytopenia. The first step is to always check a peripheral smear for clumping to rule out spurious thrombocytopenia. With a low platelet count and the absence of spurious thrombocytopenia, NAIT can be suspected. This condition is similar in pathogenesis to hemolytic disease of the fetus and newborn (HDFN), and is caused by an incompatibility in human platelet antigens between mother and baby. In about 80% of cases, the mother is found to be HPA-1b and the father and baby are HPA-1a.1 The mother forms anti-HPA-1a which crosses the placenta and destroys the fetus’ platelets. Most cases of NAIT are asymptomatic, or cause only mild bleeding, and resolve in 1-2 weeks.1

Although many cases of NAIT are mild, it is important to recognize because it can be a life-threatening disorder. With more severe thrombocytopenia, in both premature and full term infants, NAIT can result in intracranial bleeding either before birth or shortly after birth. NAIT can also cause long term neurologic complications. Therefore, when a neonate is suspected to have NAIT, he should be screened for intracranial hemorrhage. Since mothers are most often found to have anti- HPA-1a, and the second most commonly found antibody is anti-HPA-5b, neonates with platelet counts <30 x 103/L should be transfused with antigen matched or HPA-1a and HPA-5b negative, CMV negative, single donor apheresis platelets.

It is important to note that NAIT can occur in a first pregnancy but subsequent pregnancies are usually more severely affected. In confirming NAIT after a first delivery or monitoring a subsequent pregnancy, serological testing should be done on both parents to determine the risk of having an infant born with NAIT. If the father is homozygous for the antigen which the mother lacks, 100% of infants would be at risk. If the father is heterozygous, an infant would have a 50% chance of inheriting the antigen from the father.

NAIT in a first pregnancy is typically unrecognized until after birth. Some groups have advocated for routine prenatal screening for NAIT in all pregnant women, but this is costly and still debated. It is agreed that after an affected first child, subsequent pregnancies should be monitored closely. In at risk pregnancies, weekly antenatal IVIg infusions should be used during pregnancy to help prevent fetal bleeding.3

The mother in this case was tested and found to be HPA-1a negative with anti-HPA-1a. The father was also tested and found to be HPA-1a positive. The infant’s platelet counts began to increase at 7 days, with no further bleeding. The mother was referred to a NAIT specialty team for future pregnancies.

Diagnosis: Neonatal Immune Thrombocytopenia (NAIT)

  • Similar in pathogenesis to hemolytic disease of the fetus and newborn (HDFN)
  • Incompatibility in human platelet antigens between mother and baby.
  • Can affect first born
  • In majority of cases, the mother is HPA-1b and the father and baby are HPA-1a
  • Second most common is anti-HPA-5b

References

  1. http://naitbabies.org/wp-content/uploads/141208_NAIT_Registry_poster.pdf
  2. Subarna Chakravorty and Irene Roberts. How I manage neonatal thrombocytopenia . Blackwell Publishing Ltd, British Journal of Haematology. 2011; 156, 155–162
  3. T.W. de Vos, D. Winkelhorst, M. de Haas, E. Lopriore, D. Oepkes. Epidemiology and management of fetal and neonatal alloimmune thrombocytopenia. Transfusion and Apheresis Science. 2020
  4. Shamudheen Rafiyath, Immune Thrombocytopenia and Pregnancy Treatment & Management Updated: Sept. 2020 https://emedicine.medscape.com/article/208697-treatment

-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.

Truth or Consequences: The Wrong Question

It was with sadness that I watched the episode of Jeopardy! which featured Alex Trebek’s final appearance. While I hadn’t watched the game show consistently since 1984 when he first began to host, Alex had certainly become an icon in U.S pop culture and I had enjoyed watching him often. The quiz show has always been different than most- the answer must be given in the form of a question, and it must be the correct question in order to score points. As with most games, contestants don’t always ask the right question. That can happen with lab safety, as well.

I was performing an audit in a laboratory when the manager was bringing a new employee through during her orientation. I was introduced as the Lab Safety Officer, and I described some of my duties like auditing and safety compliance monitoring. The new employee immediately asked, “What happens if you catch someone not doing what they should?” That was the wrong question.

As an experienced lab safety professional, I often see people fail to follow certain lab safety regulations. Unfortunately, you do not have to look far to find lapses in lab safety practices. Vendors and service representatives and other visitors walk into labs across the country and lab staff ignore them. The visitors are not given information about the hazards in the department and they are not offered PPE. A look on social media will reveal multiple pictures of lab workers not wearing PPE as well. Oh- and they are taking those pictures with cell phones they shouldn’t be using (sometimes the hand holding the phone is gloved, other times it is not). While I am concerned about these unsafe behaviors, I am equally concerned about those that witness them and say nothing.

The COVID-19 pandemic has raised the public awareness of an important aspect of personal safety: the unsafe behavior of others can have a direct affect on your own safety. People who refuse to wear masks or who are sick and do not isolate themselves may create situations where the virus is spread to others. In the past year, many people have realized this and have felt empowered to say something to those who are not exhibiting safe behaviors. That realization that they may be in danger has made people feel comfortable speaking up for their safety and that of others around them. Perhaps that is what is needed in the lab setting as well.

Unsafe behaviors in the laboratory can easily have consequences that may affect many in the department. Spills and exposures are just some incidents that may occur. Messy lab areas can create trips or falls, and improper storage of chemicals or hazardous wastes can be dangerous as well. Perhaps laboratory staff don’t think enough about the dangerous consequences because there isn’t enough training about them. Perhaps they don’t think about the potential consequences to others because they haven’t been told about the possible physical, environmental, or financial consequences. When the new lab employee asked the question, “What happens if you catch someone not doing what they should,” I should have had an immediate answer. I should have said that she asked the wrong question. The real question is, “More importantly, what happens to you if you’re not doing what you should?” Teaching staff about the consequences of unsafe lab practices is something that should start on day one, and the awareness of these issues should be raised often and continuously. The truth is, it is important to correct your own unsafe behaviors, but it is also important to feel empowered to correct unsafe issues that are witnessed. The truth is, we all have a responsibility for our safety and that of everyone else who may be in the laboratory. If we own that responsibility, then no one’s safety has to be in…jeopardy.

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.

Eye Spy

In a previous post, I mentioned how uncontrolled cell division can occur in any cell type that has the capacity to proliferate, resulting in a neoplasm. Malignancies don’t discriminate – whether the malignant cell is identified within the skin, bladder, breast, bone, muscle, or brain – cancer is cancer. Fortunately, there are many specialists within various disciplines that can identify precancerous changes and other diagnostic abnormalities. Many patients in our head & neck clinic were referred by their dentist who identified a white patch on their tongue, a lump, or a sore that bleeds easily. Similarly, ophthalmologists can recognize ocular lesions during a routine eye exam, and I’m here to share some of those interesting ocular FNA cases. The idea of sticking a needle in your eye is perhaps one of the least pleasant thoughts a person can have, however, these FNAs are crucial for cytopathologists to analyze the cells to confirm a diagnosis.

Depending on the area of the eye sampled and the nature of the lesion, eye FNAs can yield very limited material. Optimal preservation and cytopreparation of ocular samples is imperative. At times, the sample may yield less than 50 cells, from which a diagnosis is expected to be made. Patient history and clinical impression are also crucial to properly triage the FNA. For suspected lymphomas, a portion of the sample should be sent to flow cytometry. For presumed melanomas and metastatic disease, extra preparations should be prepped for immunohistochemistry analysis. In contrast, a clinical impression of a stromal cyst is important to know as these FNAs are likely to be virtually acellular albeit rare cyst contents. The following four cases are presented with their histories and clinical impressions, supplemented by ancillary material.

Case 1. 58 year old male with a history of papillary thyroid carcinoma (2016) and renal cell carcinoma (2018). He presented with periorbital pain and a choroidal mass, OS.

Images 1-3: Eye, Left, Choroid, Fine Needle Aspiration. 1. Pap-stain; 2. vimentin +; 3. AE1/AE3 +.

Final Diagnosis: Positive for malignant cells, consistent with metastatic renal cell carcinoma.


Case 2. 58 year old female with a history of breast cancer. She presented with retinal detachment and nonpigmented nodules, OS.

Images 4-6: Eye, Left, Choroid, Fine Needle Aspiration. 4. Pap-stain; 5. GATA-3 +; 6. AE1/AE3 +.

Final Diagnosis: Positive for malignant cells, consistent with metastatic mammary carcinoma.


Case 3. 80 year old male with a history of lung adenocarcinoma (2012), pituitary adenoma (2013), and liver carcinoid (2019). He presented with a choroidal lesion, OS.

Images 7-10: Eye, Left, Choroid, Fine Needle Aspiration. 7. Pap-stain; 8. chromogranin +; 9. synaptophysin +; 10. TTF-1 +.

Final Diagnosis: Positive for malignant cells, metastatic neuroendocrine neoplasm (favor carcinoid tumor).


Case 4. 13 year old male with no cancer history. He presented with a choroidal lesion and retinal detachment, OD. Clinically suspicious for choroidal melanoma.

Images 11-14: Eye, Right, Choroid, Fine Needle Aspiration. 11. Pap-stain; 12. H&E; 13. HMB-45 +; 14. Melan A+.

Final Diagnosis: Positive for malignant cells, malignant melanoma (spindle B cell type).


Treatment of eye tumors can range from topical chemotherapies or excision for lesions on the surface of the eye, whereas intraocular tumors can be treated by brachytherapy or evisceration (removing intraocular contents and leaving the scleral shell) or enucleation (complete removal of both the intraocular contents and the scleral shell). While prosthetics are available for the latter therapies, an accurate cytopathology diagnosis is vital to guide treatment in the field of ocular oncology.

Well, that’s all that EYE have for you today. Stay tuned for next month’s cytology case study!

-Taryn Waraksa, MS, SCT(ASCP)CM, CT(IAC), has worked as a cytotechnologist at Fox Chase Cancer Center, in Philadelphia, Pennsylvania, since earning her master’s degree from Thomas Jefferson University in 2014. She is an ASCP board-certified Specialist in Cytotechnology with an additional certification by the International Academy of Cytology (IAC). She is also a 2020 ASCP 40 Under Forty Honoree.

Microbiology Case Study: A 40 Year Old Man with LVAD Exit Site Pain

Case history

A 40 year old male with a history of cardiomyopathy requiring a left ventricular assist device (LVAD) was seen in clinic with a complaint of pain at the exit site of the LVAD driveline. History is notable for multiple admissions for driveline-associated complications. Despite extensive prior evaluation, cultures and imaging of the driveline exit site had been repeatedly negative with the exception of a methicillin-susceptible Staphylococcus aureus.This was treated with nafcillin, followed by doxycycline for oral suppression. The patient had stopped taking oral antibiotics two months prior to presentation. Imaging revealed a 1.4 cm region around the driveline exit site suggestive of either phlegmon, hematoma, or a developing abscess. Blood cultures and cultures of the driveline exit site were collected and sent to the clinical microbiology laboratory. Upon physical examination, the driveline exit site was tender, but no erythema was noted. The patient endorsed intermittent rust-colored drainage from the site. Blood cultures remained negative for the duration of the patient’s hospital course, and the patient was discharged on nafcillin with plans to transition to doxycycline.

Laboratory identification

The Gram stain of the driveline exit site was unremarkable, with no organisms and few neutrophils seen. Aerobic cultures yielded a light amount of gram positive cocci in addition to mixed skin flora. Colonies were small, and weakly beta hemolytic on blood agar (Image 1A). This organism was catalase- and coagulase-positive, and definitively identified as Staphylococcus aureus by MALDI-TOF MS. Susceptibility testing was performed by broth microdilution, where the organism was determined to be a vancomycin-intermediate Staphylococcus aureus (VISA, MIC=4, Image 1C). Due to the unusual nature of the result, it repeated and confirmed by E-test (Image IB) in our laboratory, and independently verified at our contract reference laboratory. The isolate was also referred to the Texas State Public Health Laboratory where the vancomycin-intermediate phenotype was again confirmed. This isolate was also daptomycin non-susceptible, but remained susceptible to oxacillin, trimethoprim/sulfamethoxazole, linezolid, rifampin, and clindamycin.

Image 1. A. Growth of the weakly beta-hemolytic vancomycin-intermediate S. aureus strain on blood agar. B. Measurement of vancomycin susceptibility by E-test (MIC=4). C. Confirmation of the VISA phenotype by broth microdilution (MIC=4).

Discussion

All models of LVADs require a percutaneous driveline which is a link between the implanted device and the external power source.1In addition to providing power, the driveline also provides controlling and sensing functions for the LVAD.2 The driveline exit site is one of the most common sites of LVAD infection as the driveline creates a conduit for entry of bacteria from the external environment. Additionally, the prosthetic material of the driveline can serve as an ideal substrate for biofilms formation.1 The most common microorganisms associated with LVAD-related infections members of the skin microbiota (i.e. staphylococci), Pseudomonas sp., and enteric bacteria.3

Staphylococcus aureus remains an important human pathogen globally. While antibiotic intervention remains a mainstay of treatment, the emergence of resistance has historically changed the way patients are managed. Mobile genetic elements (including plasmids and transposons) are important mediators of antibiotic resistance in S. aureus, particularly with respect to beta-lactams and glycopeptide antibiotics. Due to the widespread emergence of beta-lactamase conferred penicillin-resistance, semisynthetic penicillinase-resistant penicillins (including methicillin, oxacillin, and nafcillin) were developed for clinical use in the late 1950s. However, resistance to these compounds in S. aureus was reported only a few years following their introduction. Vancomycin became the antibiotic of choice for methicillin-resistant S. aureus (MRSA) therapy in the 1980s, and contemporary management remains largely reliant on this antibiotic despite the recent availability of newer agents from different antibiotic classes.4Thus, vancomycin non-susceptibility among S. aureus isolates is a rare phenomenon with serious clinical implications, with only modest increases in vancomycin MICs resulting in treatment failures.5

The first vancomycin-intermediate S. aureus (VISA) isolate was reported in 1997 in Japan, followed by the first vancomycin-resistant isolate in 2002 in the US.4 It is important to note that the mechanisms driving these two phenotypes are entirely different. The fully vancomycin-resistant phenotype is due to the acquisition of the vanA gene which confers cell wall alterations that prohibit vancomycin from efficiently binding its target. By contrast, the vancomycin-intermediate phenotype remains less well described mechanistically, but VISA strains share similar phenotypic traits. These include: alterations in growth kinetics, increased cell wall thickness, a reduction in peptidoglycan crosslinking, decreased autolysis, altered surface protein profile, and variation of expression levels of global genetic regulators.4,5 These phenotypes are due to mutations and alterations in expression of a number of candidate genes involved in cell wall synthesis, capsule production, and global regulators of virulence.

The emergence of a VISA phenotype is usually found in the setting of MRSA strains that have been treated with prolonged vancomycin therapy.5 However, in this patient’s case, vancomycin had only been utilized infrequently for unrelated infections several years prior. Daptomycin had not previously been used in this patient’s clinical care. This VISA isolate was also oxacillin-susceptible which is a less common finding among reported VISA strains. While exposure of S. aureus to non-glycopeptide antibiotics including beta-lactams can trigger VISA phenotypes in vitro,6 it is currently not possible to elucidate the mechanism underpinning vancomycin non-susceptibility, nor what has driven this resistant phenotype, in this patient’s isolate. The patient currently is doing well on doxycycline suppressive therapy after completing his course of nafcillin, and continues to be monitored through follow-up appointments.

References

  1. Leuck A-M. 2015. Left ventricular assist device driveline infections: recent advances and future goals. Journal of Thoracic Disease 7:2151-2157.
  2. Long B, Robertson J, Koyfman A, Brady W. 2019. Left ventricular assist devices and their complications: A review for emergency clinicians. The American Journal of Emergency Medicine 37:1562-1570.
  3. Zinoviev R, Lippincott CK, Keller SC, Gilotra NA. 2020. In Full Flow: Left Ventricular Assist Device Infections in the Modern Era. Open Forum Infectious Diseases 7.
  4. McGuinness WA, Malachowa N, DeLeo FR. 2017. Vancomycin Resistance in Staphylococcus aureus
The Yale Journal of Biology and Medicine 90:269-281.
  5. Gardete S, Tomasz A. 2014. Mechanisms of vancomycin resistance in Staphylococcus aureus. The Journal of Clinical Investigation 124:2836-2840.
  6. Roch M, Clair P, Renzoni A, Reverdy M-E, Dauwalder O, Bes M, Martra A, Freydière A-M, Laurent F, Reix P, Dumitrescu O, Vandenesch F. 2014. Exposure of Staphylococcus aureus to subinhibitory concentrations of β-lactam antibiotics induces heterogeneous vancomycin-intermediate Staphylococcus aureus. Antimicrobial agents and chemotherapy 58:5306-5314.

-Zoya Khan MS, MLS(ASCP)CM is a medical technologist in the clinical microbiology laboratory at UT Southwestern with almost 10 years’ experience. She received a BS in Medical Technology from Texas Women’s University, and an MS in Clinical Practice Management from Texas Tech Health Science Center. She has an active interest in mycology and laboratory assay verification.

Francesca Lee, MD, is an associate professor in the Departments of Pathology and Internal Medicine (Infectious Diseases) at UT Southwestern Medical Center.

-Andrew Clark, PhD, D(ABMM) is an Assistant Professor at UT Southwestern Medical Center in the Department of Pathology, and Associate Director of the Clements University Hospital microbiology laboratory. He completed a CPEP-accredited postdoctoral fellowship in Medical and Public Health Microbiology at National Institutes of Health, and is interested in antimicrobial susceptibility and anaerobe pathophysiology.

Stable Chimerism Post-Double Cord Transplant

Hello again! The last case study was an example of a patient with a loss of allele at two STR loci on a shared chromosome. Today, I wanted to share an interesting and unusual case that we monitor in our lab. This case explores the use of cord bloods as the source of the donor, and in this case, a double cord blood transplant.

Cord blood (CB) unit transplants can be advantageous over other donor sources, such as bone marrow or peripheral blood. The Leukemia and Lymphoma Society summarizes these advantages well, with some being their availability (CB can be prescreened/tested and then frozen for use when needed – decreasing the risk of disease transmission), less-strict HLA matching requirements, decreased graft versus host disease (GVHD) occurrence and severity, long-term storage (CB over 10 years old has been successfully transplanted), increased diversity of donors, and reduced risk of disease relapse, to name a few.2, 3

CB also has its disadvantages, some include: less stem cells for engraftment which leads to longer engraftment times, these longer engraftment times lead to longer immunological recovery and a higher risk of infection, less available clinical data relative to stem cell and bone marrow transplants (newer procedure comparatively in transplant), and no additional cells for infusions later on in treatment. Further, selecting the best cords for transplant can be challenging due to the static variables of a CB (again, there is no donor to go back and get more cells). Considering all that CB has to offer, haplo-identical transplants are preferred in the U.S. over CB transplants. 2,3,4

Before the University of Minnesota pioneered the strategy of double cord transplants, single cord transplants gave rise to a high incidence of graft failure and transplant related mortality. 2 Double cord transplants have now become standard when utilizing CB as the donor, as a single CB unit contains a small number of required and necessary cells for a successful transplant and double units help overcome the issues that this presents.

Double cord transplants are interesting and complicated for analysis purposes (and in general!). All stem cell transplants involve a dynamic process between the cells of the donor and recipient. Yet, double cords bring in another dynamic process including an additional donor.1,2 Through the chimerism monitoring process, the complexity of the engraftment process can be appreciated as one cord ultimately becomes the “winner” and the other the “loser”. In other words, one engrafts and is detectable, while the other cord fails to engraft and becomes undetectable. Figure 1 demonstrates this process, where both cords are present initially after transplant. Then, at 43 days post-transplant, a single donor cord (D2) engrafts while the other donor cord (D1) does not engraft. D1 is most likely eliminated from the host, potentially explained by multiple theories, and no longer is detectable by chimerism testing.

Figure 1. “D1” (blue) and “D2” (pink) represent donor cord one and two alleles, respectively. “D2R” (green) represent a shared allele among donor cord two and the recipient. Each image is a time lapse of the “D18S51” STR locus post-transplant. Alleles 12, 14, 15, and 19 are present at this locus. At 21 days post-transplant, both donors are present. At 43 days post-transplant and following, only donor 2 is present and alleles 14 and 15 are no longer observed.

In the case study below, the patient was diagnosed with chronic myeloid leukemia and received a double cord transplant in 2014. One would expect, as described above, that one cord would become the “winner” while the other is rejected and becomes the “loser” and becomes undetectable. Interesting enough, this patient never achieved a status of a “winner” or “loser” cord. Rather, both remained persistent within the patient’s chimerism profile and over time have become relatively stable in their percentages.

In the electropherogram below (Figure 2), alleles from both donors can be appreciated from the CD3 (top) and CD33 (bottom) lineages. Each lineage exhibits different constitutions of the donor cord percentages, where CD3 has a greater proportion of cord two than CD33; yet both lineages have a greater overall percentage of cord two than cord one. Looking at the line graph (Figure 3), the differences between the cord percentages can be further appreciated over time. It can even be noted that the cord proportions in the CD33 lineage swapped in 2017, only to swap back to favor cord two and to remain that way since. Changes of donor-recipient relative percentages occur throughout the post-transplant journey and these events are due to complex processes. Some patients become transient mixed chimerisms (who initially are mixed chimerism but later achieve total/complete chimerism), others achieve complete chimerism, and yet others may become stable mixed chimerism. It is important to note that, even in cases where complete chimerism is not achieved, disease remission can still be present.1 In this case, the patient has achieved a stable mixed chimerism status among both donor cords and, to our lab’s knowledge, is doing well clinically.

Figure 2. “D1” (blue) and “D2” (pink) represent donor cord one and two alleles, respectively. Green D1D2R, D2R, and D1D2 represent shared alleles (where “R” represents recipient alleles). Comparing the top (CD3) and bottom (CD33) electropherograms, it can be appreciated that the percentage of each cord is different for each lineage population.
Figure 3. The red line graph on the left depicts the donor percentage of each cord blood unit (CBU) of CD3 lineage over time (11/2016 – 07/2020). It can be appreciated that CBU 2 is the dominant cord for CD3. The blue line graph on the left depicts the donor percentage of each CBU of CD33 lineage over time (11/2016 – 07/2020). It can be appreciated that CBU 2 is also dominant, but the differences between the cord donor percentages are much less compared to that of the CD3 lineage. Also, you can see over time that the two cords are relatively stabilizing in the percentages.

This case brings me back to a memory of my professor, who spoke briefly of this occurrence in a lecture only to quickly admit of its rarity. This is an interesting case because it represents one of those extremely uncommon instances. It is a privilege to be a part of a transplant center, like Northwestern’s, where we can witness rare and unique presentations like this. It opens up opportunities to learn and explore the complexities that transplant medicine and molecular HLA have to offer.

References

  1. Faraci M, Bagnasco F, Leoni M, et al. Evaluation of Chimerism Dynamics after Allogeneic Hematopoietic Stem Cell Transplantation in Children with Nonmalignant Diseases. Biol Blood Marrow Transplant. 2018;24(5):1088-1093. doi:10.1016/j.bbmt.2017.12.801
  2. Gutman JA, Riddell SR, McGoldrick S, Delaney C. Double unit cord blood transplantation: Who wins-and why do we care?. Chimerism. 2010;1(1):21-22. doi:10.4161/chim.1.1.12141
  3. Leukemia &amp; Lymphoma Society. Transplantation Facts.https://www.lls.org/sites/default/files/file_assets/FS2_Cord_Blood_Transplantation_6_16FINAL.pdf. Published May 2016. Accessed December 15, 2020.
  4. Gupta AO, Wagner JE. Umbilical Cord Blood Transplants: Current Status and Evolving Therapies. Front Pediatr. 2020;8:570282. Published 2020 Oct 2. doi:10.3389/fped.2020.570282

-Ben Dahlstrom is a recent graduate of the NorthShore University HealthSystem MLS program. He currently works as a molecular technologist for Northwestern University in their transplant lab, performing HLA typing on bone marrow and solid organ transplants. His interests include microbiology, molecular, immunology, and blood bank.

Microbiology Case Study: A 60 Year Old Male with Dysuria

Case Description

A 60 year old Hispanic male with a past medical history significant for chronic pancreatitis, hypertension and cirrhosis was admitted with decompensated cirrhosis. He underwent paracentesis for ascites and subsequently developed a hematoma as a complication of the procedure which required embolization. During his 12-day long hospital stay, he also developed hypoxia due to volume overload that improved with diuresis. A Foley catheter was placed during his hospital stay which was removed prior to discharge. Weeks later, at a follow up appointment with urology, he complained of dysuria, very little urine during voiding and the sensation of incomplete bladder emptying. A clean catch urine culture was performed and grew >100,000 colonies of Escherichia coli. As shown in Table 1, the isolate was resistant to multiple classes of antibiotics including penicillins, cephalosporins, fluoroquinolones, one aminoglycoside (Tobramycin), Trimethoprim/Sulfamethoxazole, aztreonam and carbapenems (Ertapenem/Meropenem) making this isolate multi-drug resistant (MDR). Because of the resistance profile to the carbapenems, molecular testing for carbapenemase genes was performed and the New Delhi metallo-beta-lactamase (NDM-1) gene was detected. The patient was treated with nitrofurantoin for his symptomatic urinary tract infection (UTI).

Table 1. Antimicrobial susceptibility of this isolate of E. coli.

Discussion

Escherichia coli is a gram negative, motile bacillus that is a normal constituent of the gastrointestinal tract and is one of the most common causes of uncomplicated UTI. Antimicrobial susceptibilities are nearly always performed because the isolates of E. coli can vary in resistance. E. coli do not have any intrinsic resistance to antibiotics other than penicillin; however, they can acquire resistance through numerous mechanisms including structural mutations and plasmid-borne genes that encode enzymes to various classes of antibiotics. One such plasmid-encoded enzyme is the NDM, which was identified in our patient’s isolate. Its resistance is the result of bacterial synthesis of a carbapenemase that deactivates carbapenems by breaking down the beta-lactam ring.1 In the United States, K. pneumoniae carbapenemase (KPC) is the most common, but other types carbapenemase enzymes have also been reported.1,2 NDM is uncommonly isolated in E. coli; it is more often identified in other gram negative bacteria including MDR Pseudomonas aeruginosa or Acinetobacter baumannii complex, which can cause, among other things, devastating nosocomial infections within a healthcare setting. Because these enzymes are on mobile elements, a patient can be colonized with one bacterial strain that carries the plasmid with the carbapenemase on it and transfer a copy of that plasmid to another bacterial strain, thereby conferring new carbapenem resistance to the new bacterium (e.g., P. aeruginosa with the NDM on a plasmid shares that plasmid with an E. coli). Carbapenem resistant Enterobacteriaceae (CRE) are of great importance in healthcare. Carbapenem resistance mediated by enzyme activity (e.g., KPC, NDM, OXA-48, etc), typically confers resistance to all beta lactams. Interestingly, NDM enzymes typically do not destroy aztreonam, a monobactam;3 however, it is common for bacteria to have multiple resistance genes, so NDM carrying strains can be resistant to aztreonam. Although these CRE isolates can cause significant morbidity and mortality when found in clinical samples including sputum or blood, luckily for our patient, he had an uncomplicated UTI and nitrofurantoin was susceptible.

-Limin Yang is a PGY-1 resident in Anatomic and Clinical Pathology at University of Texas Southwestern. She has varied interests including anatomic pathology specialties.

-Dominick Cavuoti is a professor of Anatomic and Clinical Pathology at UT Southwestern and active faculty on both Microbiology and Cytology services.

-Clare McCormick-Baw, MD, PhD is an Assistant Professor of Clinical Microbiology at UT Southwestern in Dallas, Texas. She has a passion for teaching about laboratory medicine in general and the best uses of the microbiology lab in particular.

Will the B.1.1.7 variant evade the Vaccine/Tests?

Will the B.1.1.7 variant evade the vaccine/tests?

This question came up recently and I wanted to share some cutting edge information the addresses this. This was in part adapted from Akiko Iwasaki’s (Yale HHMI immunologist) Twitter discussion of this subject.1

Will B.1.1.7 evade our tests?

The UK variant commonly called lineage B.1.1.7 (officially Variant of Concern 202012/01) has 23 genetic variants that result in 17 protein coding changes.2 Most tests including the ones at our institution (Abbott) are not currently affected (see below). Only the ThermoFisher assay has declared a target that covers the 69-70del variant in the S gene (in green). This conversely makes the TaqPath® assay one way to detect a potential B.1.1.7 variant.

Figure 1. A picture of the SARS-CoV-2 genome with red lines indicating mutation sites and different assays and relative location of their qPCR targets.

Will the vaccine protect against the B.1.1.7 variant?

The Pfizer and Moderna RNA vaccines create an immune response against the spike protein. We don’t know the exact sequences or reactivity of the vaccines’ spike protein. However, a recent study looked at the antibody reactivity to linear epitopes of COVID-19 in 579 patients who were naturally infected with COVID-19. For the antibodies against the spike, the major reactive linear epitopes are indicated in Red at the bottom. None of the B.1.1.7 mutations (Orange) overlap with these major reactive epitopes.3 

Figure taken from Reference 3.

For a closer look, see below.

Figure taken from Reference 3.

A limitation of these analyses is the use of only linear epitopes. Mutations might impact a 3D epitope affecting Ab binding. However, people make multiple antibodies to the spike protein.4 So, broad coverage should arise after exposure to the either the vaccine or natural infection with COVID-19.

The vaccine should induce a polyclonal antibody response that recognizes multiple parts of the spike protein, making it effective, even against novel variants. Also, there should be few to no False Negative COVID-19 tests due to the new variant, but we will continue to monitor and test this experimentally. 

References

  1. Prof. Akiko Iwasaki @VirusesImmunity
  2.  Chand, Meera et al. Investigation of novel SARS-COV-2 variant: Variant of Concern 202012/01 Public Health England.
  3. Haynes WA et al. High-resolution mapping and characterization of epitopes in COVID-19 patients. MedRxiv. https://www.medrxiv.org/content/10.1101/2020.11.23.20235002v1#p-5
  4. Shrock E et al. Viral epitope profiling of COVID-19 patients reveals cross-reactivity and correlates of severity. Science 2020 370(6520). https://science.sciencemag.org/content/370/6520/eabd4250

Jeff SoRelle, MD is Assistant Instructor of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX working in the Next Generation Sequencing lab. His clinical research interests include understanding how lab medicine impacts transgender healthcare and improving genetic variant interpretation. Follow him on Twitter @Jeff_SoRelle.

Cervical Cancer Prevention: The Disparate Reality Across the Globe

One of the most effective public health strategies to date is the development of the pap smear test and its use as a screening tool in cervical cancer prevention. Before the emergence of the pap test, cervical cancer used to be the leading cause of cancer-related deaths for women in the United States.1 However, with effective pap tests screening programs and the availability of the HPV vaccine against high-risk serotypes known to be a major cause of cervical cancer, many developed countries have been able to address this disease with its attendant catastrophic consequences including loss of lives, income and long-term disabilities.

Now, cervical cancer doesn’t even make it as one of the top 10 causes of cancer-related deaths for women in the United States.2 Unfortunately, the progress that has been made with this disease is not a universal one. While many developed countries have made giant strides in addressing this disease burden, most developing countries still grapple with significant morbidities and mortalities attributable to cervical cancer. Recent statistics show that cervical cancer is the second most commonly diagnosed cancer after breast cancer and the third leading cause of cancer death after breast and lung cancers in developing countries.3 In fact, almost 90% of cervical deaths in the world occur in developing countries, with India alone accounting for 25% of the total cases.3 Cervical cancer incidence and mortality rates are highest in sub-Saharan Africa, Central and South America, South-eastern Asia, and Central and Eastern Europe.3 A combination of factors may be responsible for these discrepant findings in developing countries.

First is the lack of effective screening programs that detect precancerous lesions before they become invasive diseases. Unfortunately, this factor is linked to lack of awareness through education and sub-optimal laboratory services that still exists in many of these countries. Laboratory services are scarce and there has been a gradual decline in laboratory professionals. Even if the supplies and equipment needed to run a lab were available, where are the laboratory professionals and pathologists that are needed to provide this critical healthcare service? In addition, a lack of regulatory oversight in some of these countries makes the replication and standardization of results increasingly challenging.

Secondly, is the role of HPV vaccinations in preventing cervical cancer. Many of these developing countries are yet to incorporate routine HPV vaccinations into their vaccination programs and access to these services are still very low. In contrast, many developed countries have made HPV vaccinations available and accessible, which is a major defense against cervical cancer.

Thirdly, is the impact of government policies on laboratory medicine. Pathology and laboratory medicine continue to face cuts in services and compensations, even in developed countries including the United States. These practices impact the ability of laboratory services to deliver optimal results, a scenario that could be even more problematic in developing nations.

As January marks cervical cancer awareness month, public health and policy professionals need to take steps to address the root causes of this problem, in order to proffer sustainable solutions, especially in developing countries. In addition to prioritizing health education and public health campaigns on cervical cancer prevention, the role of effective laboratory services in addressing these challenges also need to be emphasized.

A successful healthcare initiative requires a strong and functioning laboratory system, especially in the 21st century. Any health policy program or public health campaign that fails to recognize this fact is most likely headed for failure before it even starts off.

References

  1. https://www.cdc.gov/cancer/cervical/statistics/index.htm
  2. https://www.cancer.org/research/cancer-facts-statistics/all-cancer-facts-figures/cancer-facts-figures-2020.html
  3. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. Lyon, France: International Agency for Research on Cancer. Available from: http://globocan.iarc.fr, 2013.

-Evi Abada, MD, MS is a Resident Physician in anatomic and clinical pathology at the Wayne State University School of Medicine/Detroit Medical Center in Michigan. She earned her Masters of Science in International Health Policy and Management from Brandeis University in Massachusetts, and is a global health advocate. Dr. Abada has been appointed to serve on the ASCP’s Resident’s Council and was named one of ASCP’S 40 under Forty honorees for the year 2020. You can follow her on twitter @EviAbadaMD.