Global Health Narratives Interview Series: Meet Dr. Von G. Samedi

Von G. Samedi, MD, PhD, is a cytopathologist at the University of Colorado in Denver, CO. I had the pleasure of meeting Dr. Samedi as a result of the thoughtful introduction facilitated by Dr. Melissa Upton, who thought we should talk given our shared interest in global pathology.

I learned that Dr. Samedi is originally from Haiti and completed his MD, PhD, and pathology training in the US. He has always been interested in global health as part of his personal and professional passion and has spent the last decade dedicating his expertise to improving pathology services in low resource settings. It was readily apparent to me that Dr. Samedi’s approach to the world’s healthcare issues is based in the fact that he views these as shared problems – ones that he can and does help solve. This mindset is reflected in the way he lives his life – admirably contributing to society in any way that he possibly can. I was eager to hear of the opportunities he’s found in order to contribute, so that I might learn and share with all of you the ways that we can all get involved. Read on to discover the inspiring story of someone who has persisted in finding ways to give to the world through service!

Q: When did you first get started working in global health through pathology?

A: I started working with ASCP when I was a 4th year pathology resident in 2010 when they called me to assist their project in Haiti, which was in response to the tremendous damage caused from the earthquake. I had signed up as a potential volunteer on their website prior to this and they reached out to me seeing that I had language proficiency in both French and Creole. I spent 21 days working with them and my residency program allowed me to count this time as an outside elective. Their main goal was to work with the Haiti’s national public health laboratory (Laboratoire National de Santé Publique) and its various national and international partners to set up and run a laboratory in this acute disaster situation, and the hands-on experience I gained in doing this was well worth my program elective time.

After this, ASCP requested that I continue to volunteer with them and since then, I have been working on pathology and laboratory medicine improvement projects at their partner sites all over the world.

Q: Can you tell me about your experiences volunteering with ASCP’s global health initiatives?

A: Working with ASCP at their global partner sites has allowed me to volunteer in a variety of ways which is unique to the needs of each situation. Every trip has been different. In Botswana, I helped process and read the cervical biopsy specimens that had accumulated as a result of a government program to address the high incidence of cervical cancer. The biopsy program was successful except that there weren’t enough pathologists to give results from the tissue samples – so the government reached out to ASCP to help fill the gap in care. In Ukraine, I worked with laboratorians and clinicians in which I helped conduct a workshop on HIV related testing services. In the Ivory Coast, I worked as a part of a mentorship program to assist a newly formed pathology organization gain functional independence. In Rwanda, the project was focused on bringing telepathology services into the laboratory. In Kenya, I worked with ASCP to offer support to the local pathology association. I’ve also returned to Haiti since 2010 and now we’ve shifted away from disaster management and focused on local laboratorian training with the goal of achieving sustainability.

Q: Why do you volunteer to improve global pathology services?

A: Historically, pathology and global health are not thought of as connected, yet without pathology, there is no practice of modern medicine. It is the same anywhere in the world as it is in the US, you must have a functioning pathology laboratory in order to effectively deliver health care. Once you understand this, you understand the need that exists in low- and middle-income countries where there is ample opportunity to serve and give back. Doing so gives me a sense of purpose and it is not just a one-way relationship, as I also benefit from interacting with my global colleagues and learning from them. What I have seen my colleagues do with so few resources is impressive and inspiring.

Q: How do you fit volunteering into your schedule?

A: My volunteering experiences have ranged anywhere between 3 to 21 days. I prioritize this work and have been fortunate to work for departments that support it, often allowing me to use professional time and vacation time to work on these projects.

Q: What advice would you give someone new to engaging in global health?

A: The key is to focus on building relationships for the long term. Be patient, flexible, and realize that what you want to accomplish may not happen in the first or even the second visit. Sometimes things just don’t go as planned and you have to keep working and go with the flow. If anyone in laboratory medicine is looking for volunteering opportunities, reach out to ASCP and volunteer to get involved – you can travel to their partner sites, volunteer to read cases through their telepathology program, or serve on ASCP’s global health committees. There’s a way for everyone and anyone working in laboratory medicine to get involved, no matter what your specialty and capacity to serve is.

-Dana Razzano, MD is a former Chief Resident in her fourth year in anatomic and clinical pathology at New York Medical College at Westchester Medical Center and will be starting her fellowship in Cytopathology at Yale University in 2020. She is passionate about global health and bringing pathology and laboratory medicine services to low and middle income countries. She was a top 5 honoree in ASCP’s Forty Under 40 in 2018 and was named to The Pathologist’s Power List of 2018 and 2019. Follow Dr. Razzano on twitter @Dr_DR_Cells.

The Best Gift of the Season: A Gift of Self

We few, we happy few, we band of brothers;

For he today that sheds his blood with me

Shall be my brother.

Henry V (Act 4, scene 3), Shakespeare.

Good morning! We’re entering the holiday season, and it’s an exciting time for all. I love seeing the ethnic and cultural diversity as we all celebrate our favorite holidays with family and friends. I myself look forward to the holiday season. It’s a festive time and a season of giving and sharing. It’s a favorite time of year to share traditions and create new ones. However, at a time when stores have Christmas candy on the shelves, holiday lights up and holiday music playing on the day after Halloween, I feel a bit rushed and want to slow down and find better ways to celebrate and enjoy the season. Over the past few years I have been making a special effort to become more environmentally conscious; remembering my reusable bags at stores, purchasing more reusable products, and reusing, recycling, and upcycling whenever I can. I belong to a community ‘buy nothing’ group and am warmed by the generosity of strangers to others in the community. It’s wonderful to give from our abundance and to receive wish list items from neighbors without having to exchange money. And it’s great for the environment, too. Used items are being put to use by others, and not into landfills. People in the community have asked for or gifted furniture, clothing, tools, toys and many other goods and services. I have gifted no longer needed clothing, household items, excess fabric from my fabric stash, and donated my time to participate in a career fair at a local high school. I have been given a car set for my grandchildren when they visit, toys, and someone even loaned me a bike trailer so we could take my granddaughter out for a bike ride. The generosity makes it feel like the holiday season all year round.

So, you may be asking, “where is this blog going?” I saw a memo from Red Cross this week that there is a critical need for blood and platelets and thought that giving to our community with the gift of blood would be a wonderful way to make this holiday season even better! It’s one of the most generous gifts we can give, and costs nothing. Every 2 seconds in the US, someone needs a blood product. That’s about 36,000 units of red blood cells, 7,000 units of platelets and 10,000 units of plasma needed every day. 21 million blood products are transfused every year.1 That’s a lot of blood. And, these blood products cannot be manufactured, so must come from volunteer donors.

In the US, we need to collect about 13,000 units a day to meet demand. Approximately 14 million units of whole blood are collected each year from roughly 7 million donors.1 The blood is processed into components and used in the treatment of surgical, obstetric, oncology, and other patients. One unit of whole blood can be made into up to 3 components and used to help up to 3 patients. Yet, even with all these donations we still cannot keep up with demand. Weather, holidays, illness and travel can all affect blood donations. Shortages are not just apparent during the winter holiday season. This past summer, the Red Cross announced a critical blood shortage around the July 4th holiday. Compared to other weeks, there were 17,000 fewer blood donations during the week of July 4th. As of July 9, the Red Cross had less than a three-day supply of most blood types and less than a two-day supply of Type O blood. 2 During the summer, and particularly during the holiday week, people are busy with other activities or traveling. In the winter, busy schedules, holiday travel, winter weather and seasonal illnesses contribute to fewer blood and platelet donations. Severe weather can also cause the cancellation of blood drives which greatly impact the blood supply.

Some people donate blood because they see this critical need and hear the calls for blood. Others donate because a classmate or friend asked them to. Some people feel it’s their civic duty. For some, it just makes them feel good to help another person. And, others donate for the cookies and tee shirt. Yet, for all donors, it is a form of volunteerism and giving to the community. But, did you know that, other than the benefits from helping others, there are benefits to the donor, as well? Helping others can improve our emotional and physical health. It can help reduce stress, improve emotional well-being and help people feel a sense of belonging. A study conducted in Sweden concluded that regular blood donors enjoy better than average health.Blood donors had an overall mortality 30% lower and a cancer incidence 4% lower than the control population.3 Donating blood may help reduce high iron stores, a risk factor for heart attack. In addition, there have been several studies over the past few years, exploring the hypothesis that regular blood donations may help in the management of hypertension and high cholesterol.

Another interesting benefit of blood donation is being able to contribute to science and research. For example, there is currently a study being conducted on donor blood to test an investigational nucleic acid test for Babesia microti. Babesia microti is responsible for most transfusion-transmitted babesiosis cases in the United States, but there is no licensed test for screening for B. microti in donated blood. Participation in this study can help obtain FDA approval for a screening test. By giving your consent to use your blood sample, there is no additional blood taken and no further time commitment, but you can help protect the public health by supporting the development of a new blood safety test.

How can we, as individuals, help? About 38% of the population is eligible to donate blood, but less than 10% of the population actually donates. To be eligible to donate, you should be in good general health and feeling well. You must be at least 17 years old in most states (16 years old with parental consent in some states) but there is no age limit to donation. Adult doors must weigh 110 lbs, but there are additional height and weight requirements for donors 18 years old and younger. There have also been some recent changes to blood donor requirements. I will not be able list all of them here, but some of them don’t change a deferral, only the reasoning behind the deferral. One of the most prominent changes is, as of 2016, the indefinite deferral for men who have had sex with men, has been changed to a 12 month deferral since the last sexual contact with another man . Also changed is the minimum hemoglobin for male donors. This has been raised from 12.5g/dl to 13.0 g/dl. Until this time, the cutoff was the same for both males and females. Males with a Hgb below 13.0 g/dl are considered anemic and are no longer eligible to donate blood. On the other hand, the criteria for females to be mildly anemic is a Hgb below 12.0 g/dl, so females between 12.0 g/dl and 12.5 g/dl, though not considered anemic, are still not eligible to donate. The minimum hemoglobin for females has not changed and remains 12.5 g/dl. To review other eligibility requirements, visit https://www.redcrossblood.org/donate-blood/how-to-donate/common-concerns/first-time-donors.html

So, in this busy season, we often find ourselves with little time to get our own “to do” lists done, yet alone volunteer our time for others. But most of us would welcome an hour to reduce stress and improve our emotional well-being. Please consider a gift of self this season. It takes about an hour of your time, you get to sit and relax with your feet up, to feel good about yourself, and you’ll even get a snack!

Happy Holidays!

References

  1. redcrossblood.org
  2. https://news.azpm.org/p/news-splash/2019/7/19/155196-fourth-of-july-donation-slowdown-leads-to-blood-shortage/
  3. Edgre, G et al. Improving health profile of blood donors as a consequence of transfusion safety efforts. Transfusion. 2007 Nov;47(11):2017-24.
  4. Kamhieh-Milz S, et al.Regular blood donation may help in the management of hypertension: an observational study on 292 blood donors. Transfusion. 2016 Mar;56(3):637-44. doi: 10.1111/trf.13428. Epub 2015 Dec 8.

-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.

Vitamin D: An Overview

Introduction

Vitamin D is one of the most commonly ordered laboratory tests in the primary care setting, as well as one of the most widely used forms of dietary supplementation today. While the rationale underlying vitamin D testing and supplementation for deficiency may seem straightforward, in actuality, the metabolism and physiologic functions of vitamin D in the body are quite nuanced and complex, and there remains significant controversy surrounding the appropriate utilization of vitamin D measurement and clinical interpretation of vitamin D test results. In this post, let’s review the basic principles of vitamin D metabolism, its function and mechanisms of regulation in the human body, methods of measurement in the laboratory, and ramifications of vitamin D values on clinical decision-making and management.

Vitamin D Metabolism

Vitamin D is a fat-soluble vitamin and encompasses a group of compounds, all containing a four-ring steroid backbone. The two main forms of vitamin D utilized by humans are vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Vitamin D3 is primarily synthesized in the skin from 7-dehydrocholesterol in the presence of sunlight (UVB rays), while vitamin D2 is synthesized in plants from ergosterol and is used to fortify many foods (milk, bread, cereal, etc.).1

Once synthesized in the skin or ingested from the gastrointestinal tract, both vitamin D2 and vitamin D3 travel in the bloodstream (bound to vitamin D-binding protein) to the liver, where both are converted to 25-hydroxyvitamin D [25(OH)D, calcidiol/calcifediol] by the action of 25-hydroxylase.1,2 While 25(OH)D has only limited biologic activity, it has a very long half-life (2-3 weeks) and is therefore the primary form of vitamin D found in the blood.1 Notably, the half-life of 25(OH)D2 is shorter than that of 25(OH)D3 (possibly due to lower affinity to vitamin D-binding protein) and therefore it is present in significantly lower concentrations than 25(OH)3 in the blood.3

25(OH)D is then further converted to 1,25-dihydroxyvitamin D [1,25(OH)2, calcitriol] via the action of 1-α-hydroxylase primarily in the kidney.1 In contrast to 25(OH)D, 1,25(OH)2D is the biologically active form of vitamin D, but it has a much shorter half-life (5-8 hours) and therefore has much lower circulating levels in the blood.1 25(OH)D may alternatively be converted to 24,25-dihydroxyvitamin D [24,25(OH)2D] by 24-α-hydroxylase, also in the kidney. 24,25(OH)2D is an inactive metabolite and thus serves as an end-product in this degradation-type pathway of 25(OH)D.1,4

Vitamin D Physiology

The overall effect of vitamin D in the body is to increase calcium and phosphate levels in the blood. Via binding of 1,25(OH)2D to nuclear receptors within cells, it acts at three main sites: 1) the intestine, where it increases calcium and phosphate absorption, 2) the bones, where it increases calcium resorption by promoting osteoclast maturation, and 3) the kidney, where it increases calcium reabsorption by enhancing the effects of parathyroid hormone (PTH) on the distal convoluted tubule.1

Conversion of 25(OH)D to 1,25(OH)2D by 1-alpha-hydroxylase is tightly regulated by calcium, phosphate, and PTH concentrations in the body. Decreased calcium or phosphate levels, or increased PTH levels in the blood (most commonly resulting from a fall in calcium) will stimulate 1-α-hydroxylase activity and lead to increased production of 1,25(OH)2D, while increased calcium or phosphate levels or decreased PTH levels will suppress 1-α-hydroxylase activity and thus lead to decreased production of 1,25(OH)2D.1,2

From a clinical perspective on vitamin D physiology, there are numerous causes of abnormal vitamin D levels in the body. Here are some common causes of low vitamin D levels:

  • Inadequate intake of vitamin D (whether from diet, inadequate sunlight, or malabsorption)
  • Decreased PTH (hypoparathyroidism, hyperphosphatemia, hypercalcemia of malignancy)
  • End-organ resistance to PTH (pseudohypoparathyroidism)
  • Decreased 1-α-hydroxylase activity (renal failure, vitamin D-dependent rickets type 1)5

Conversely, causes of high vitamin D levels are listed below:

  • Excessive intake of vitamin D (usually from supplements)
  • Increased PTH (primary hyperparathyroidism)
  • Increased extrarenal 1-α-hydroxylase activity (seen in granulomatous diseases such as sarcoidosis, as well as some lymphomas)
  • End-organ resistance to vitamin D (vitamin D-dependent rickets type 2)5

Vitamin D Measurement in the Laboratory

25(OH)D is the most commonly measured vitamin D metabolite in laboratory assays, since (as mentioned above) it has a longer half-life and a larger concentration in the blood compared to 1,25(OH)2D. In addition, its concentration does not fluctuate as significantly as that of 1,25(OH)2D, since its production from 25-hydroxylase in the liver is not so tightly regulated as 1-α-hydroxylase activity in the kidney.1,6 Nevertheless, 1,25(OH)2D measurement is indicated in a few specific clinical circumstances, including workups for idiopathic hypercalcemia and bone/mineral disorders, and for evaluation of vitamin D status in the setting of renal failure (where 1-α-hydroxylase activity is decreased).1,7

While the gold standard for vitamin D measurement is liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), most laboratories utilize immunoassays (including radioimmunoassays, chemoluminescent immunoassays, and enzyme-linked immunoassays) for vitamin D quantitation.1,7 One significant difference between these two methods is that while LC-MS/MS can differentiate vitamin D3 and vitamin D2 metabolites, immunoassays cannot.6 In addition, the antibodies used in many 25(OH)D immunoassays often have lower cross-reactivities with 25(OH)D2 and therefore may underestimate this form when giving the total 25(OH)D value.6 These antibodies also have varying cross-reactivities with other vitamin D metabolites and so may result in an overestimation of the total 25(OH)D due to positive interference from these metabolites.6

Another advantage of the LC-MS/MS method is that it can detect C3 epimers of 25(OH)D, while immunoassays cannot.8 The physiologic significance of these epimers has not yet been clearly delineated, but recent evidence has shown that while these epimers do not affect calcium concentrations, they do contribute to suppression of PTH levels.8 In addition, while these epimers comprise a low proportion (about 2-3%) of the overall 25(OH)D concentration in adults, they have been found in significantly higher proportions (up to 60%) in infant and pediatric populations.8,9 Thus, the detection of these epimers (and their quantitation, which is possible through high-performance LC-MS/MS) may be more important in these patient populations.

Interpretation of Vitamin D Results

The optimal serum levels of 25(OH)D are not universally established. First of all, levels vary with factors affecting sunlight exposure including latitude, skin pigmentation, and sunscreen use.1 Levels also demonstrate significant seasonal variation, with winter measurements up to 40-50% lower than summer measurements.1 Recommended minimum 25(OH)D levels for optimal bone health differ among various national organizations and generally range from 20 ng/mL to 30 ng/mL; as mentioned above, these thresholds are controversial and there is no established consensus.10-12

Vitamin D deficiency is very common, with the majority of patients exhibiting no clinical symptoms and normal calcium and phosphate concentrations. However, a significant proportion of these asymptomatic patients will have increased PTH levels and concomitant increased risk of osteopenia/osteoporosis and fractures; therefore, all patients with vitamin D deficiency should be treated with repletion.13 If deficiency is severe and persistent, bone demineralization with rickets (in children) and osteomalacia (in adults and children) can develop. In contrast, vitamin D toxicity is very rare and is usually associated with over-supplementation; patients develop hypercalcemia with related symptoms including confusion, muscle weakness, nausea and vomiting, and polydipsia and polyuria.14

Recent studies have linked vitamin D deficiency (usually with residency at higher latitudes) to a wide variety of clinical disorders ranging from autoimmune diseases (multiple sclerosis, rheumatoid arthritis, type I diabetes), to cancers (including colon, breast, and prostate), to psychiatric illnesses (schizophrenia, depression), and cardiovascular disease (including hypertension and congestive heart failure).15 Whether these links possess a causal basis or are merely associative needs to be further investigated. Nevertheless, what is certain is that understanding the functions of vitamin D in the body and methodologies of vitamin D measurement in the laboratory is crucial in appreciating its clinical significance and various, ever-expanding applications in disease pathophysiology and management.

References

  1. McPherson RA, Pincus MR. Henry’s Clinical Diagnosis and Management by Laboratory Methods. Elsevier Health Sciences; 2017.
  2. Brown AJ. Regulation of vitamin D action. Nephrology, dialysis, transplantation: official publication of the European Dialysis and Transplant Association-European Renal Association. 1999 Jan 1;14(1):11-6.
  3. Armas LA, Hollis BW, Heaney RP. Vitamin D2 is much less effective than vitamin D3 in humans. The Journal of Clinical Endocrinology & Metabolism. 2004 Nov 1;89(11):5387-91.
  4. Cashman KD, Hayes A, Galvin K, Merkel J, Jones G, Kaufmann M, Hoofnagle AN, Carter GD, Durazo-Arvizu RA, Sempos CT. Significance of serum 24, 25-dihydroxyvitamin D in the assessment of vitamin D status: a double-edged sword?. Clinical chemistry. 2015 Apr 1;61(4):636-45.
  5. Clarke W. Contemporary practice in clinical chemistry. Amer Assn for Clinical Chemistry; 2016.
  6. Zerwekh JE. Blood biomarkers of vitamin D status. The American journal of clinical nutrition. 2008 Apr 1;87(4):1087-91.
  7. Hollis BW. Assessment and interpretation of circulating 25-hydroxyvitamin D and 1, 25-dihydroxyvitamin D in the clinical environment. Endocrinology and Metabolism Clinics. 2010 Jun 1;39(2):271-86.
  8. Lutsey PL, Eckfeldt JH, Ogagarue ER, Folsom AR, Michos ED, Gross M. The 25-hydroxyvitamin D3 C-3 epimer: distribution, correlates, and reclassification of 25-hydroxyvitamin D status in the population-based Atherosclerosis Risk in Communities Study (ARIC). Clinica chimica acta. 2015 Mar 10;442:75-81.
  9. Singh RJ, Taylor RL, Reddy GS, Grebe SK. C-3 epimers can account for a significant proportion of total circulating 25-hydroxyvitamin D in infants, complicating accurate measurement and interpretation of vitamin D status. The Journal of Clinical Endocrinology & Metabolism. 2006 Aug 1;91(8):3055-61.
  10. Del Valle HB, Yaktine AL, Taylor CL, Ross AC, editors. Dietary reference intakes for calcium and vitamin D. National Academies Press; 2011 Apr 30.
  11. Vieth R. What is the optimal vitamin D status for health?. Progress in biophysics and molecular biology. 2006 Sep 1;92(1):26-32.
  12. American Geriatrics Society Workgroup on Vitamin D Supplementation for Older Adults. Recommendations abstracted from the American geriatrics society consensus statement on vitamin D for prevention of falls and their consequences. Journal of the American Geriatrics Society. 2014 Jan;62(1):147-52.
  13. Valcour A, Blocki F, Hawkins DM, Rao SD. Effects of age and serum 25-OH-vitamin D on serum parathyroid hormone levels. The Journal of Clinical Endocrinology & Metabolism. 2012 Nov 1;97(11):3989-95.
  14. Ozkan B, Hatun S, Bereket A. Vitamin D intoxication. Turk J Pediatr. 2012 Mar 1;54(2):93-8.
  15. Holick MF. Vitamin D deficiency. New England Journal of Medicine. 2007 Jul 19;357(3):266-81.

-Michelle Lin, MD, is a second-year anatomic and clinical pathology resident at Houston Methodist Hospital in Houston, Texas.

Personalized Medicine and Precision Medicine

There are often new buzzwords flying around that everyone uses, but few actually understand what they mean. Personalized and precision medicine are two of these terms that are often used interchangeably. Every lab wants to say they are performing personalized medicine. And to be fair we really do all provide personalized medicine in some form. Almost all lab results are used to customize the treatment for patients. However these buzzwords are used to refer to tests that describe linking genetic, lifestyle, or environmental information with predicted response to treatment. Precision medicine may be the more accurate term to describe identifying effective treatment for the right patient at the right time based on genetic, lifestyle, or environmental information. The term personalized medicine may give the false impression that therapies were developed specifically for the patient, when really they are developed to target a specific genotype or phenotype.

One example of precision medicine being used clinically today is in oncology. Many cancer drugs now require an associated test to determine the presence or absence of a specific biomarker to determine which patients are likely respond to the therapy. The biomarker tests that are linked to a specific therapy are called companion diagnostics. Biomarkers analyzed can be a specific protein or gene such as programmed death ligand-1 (PD-L1) or epidermal growth factor receptor (EGFR) or they can be much broader such as tumor mutational burden (TMB) or immune signatures. Identifying biomarkers that determine which patients are likely to respond to therapy and only giving patients with the biomarker the drug increases response rates to the therapy and may decrease side effects. More than half of the clinical trials for cancer drugs in 2018 were linked to a specific biomarker. Linking drug selection with specific laboratory tests is causing an increased need for multidisciplinary collaboration among pathology, oncology, and the laboratory.

In our lab we perform precision medicine using PCR or NGS assays to analyze patient’s tumor for specific genes. Although we still perform single gene testing when ordered, most of our cases are analyzed by a NGS panel. NGS panel testing allows us to look at numerous biomarkers with one test. This decreases the cost, time and tissue utilized to determine the patient’s biomarker status. Our NGS panel analyzes 52 genes to look for mutations that would indicate a patient is likely to respond to a targeted therapy. Most of our oncology testing is done on lung, colon, and melanoma specimens, although the panel is validated for most solid tumors. The report that we issue the oncologist provides clear information on which therapies the patient is likely to respond to or likely to be resistant to based on their tumor’s genetic profile. We also include information in the report to match patients to clinical trials. Precision medicine utilizing panel NGS testing for predicted response to treatment is becoming standard of care for many solid tumors.  

-Tabetha Sundin, PhD, HCLD (ABB), MB (ASCP)CM,  has over 10 years of laboratory experience in clinical molecular diagnostics including oncology, genetics, and infectious diseases. She is the Scientific Director of Molecular Diagnostics and Serology at Sentara Healthcare. Dr. Sundin holds appointments as Adjunct Associate Professor at Old Dominion University and Assistant Professor at Eastern Virginia Medical School and is involved with numerous efforts to support the molecular diagnostics field. 

Microbiology Case Study: A Novel Anaerobic Pathogen Causing Septic Arthritis

Clinical history

A 65 year old man with diabetes mellitus type 2 presented to the emergency department (ED) for left hip pain. He has a remote history of avascular necrosis of bilateral hips of unknown etiology for which he received a bilateral total hip arthroplasty and subsequent multiple revisions due to hardware failure several years ago. He initially presented to an urgent care clinic a few months prior for “noise with movement” of the left hip and mild lower back pain. Plain radiographs of the left hip in comparison to his prior imaging were unremarkable and he was subsequently discharged. Repeat imaging at a follow-up visit at the orthopedic clinic showed mild superior migration of the femoral head bilaterally secondary to periprosthetic osteolysis of the joint headliner. He was scheduled for surgery however presented to the ED prior to his scheduled appointment with severe crushing left hip pain and restricted joint mobilization. He denied fevers, chills, night sweats, or any other recent infections. The left hip was aspirated yielding 10cc of dark black fluid and a stat gram stain was ordered.

Laboratory identification

The stat gram stain showed many polymononuclear cells with moderate gram positive bacilli in a background of dark inorganic material (Image 1). Following 48 hours of incubation, there was anaerobic growth on the kanamycin and vancomycin (KV) and schaedler agar plates. A Gram stain of the broth showed gram positive bacilli arranged singly and in chains with some decolorization (Image 2). The KV and schaedler plates showed moderate growth of a single organism consisting of small glossy tan colored colonies (Images 3-4). No aerobic growth was observed on the blood, MacConkey, Columbia Naladixic Acid (CNA), or chocolate agar plates. Mass spectrometry (MALDI-TOF) identified the pathogenic organism as Clostridium innocuum.

Image 1. Synovial fluid Gram stain of the left hip showed moderate gram positive bacilli and many polymononuclear cells in a background dark inorganic debris (100x oil immersion).
Image 2. Gram stain from a positive broth culture showed gram positive bacilli arranged singly and in chains with some decolorization (100x oil immersion).
Image 3. Anaerobic growth on the schaedler agar showed growth of a single organism consisting of small round glossy tan colored colonies.
Image 4. Anaerobic growth on the kanamycin and vancomycin (KV) agar showed growth of a single organism consisting of small glossy tan colored colonies.

Discussion

Bacterial joint infections are more common in prosthetic joints as compared to native joints with a prevalence of 1-2% following hip arthroplasty (1). Most cases of bacterial septic arthritis are due to staphylococci (40 percent), streptococci (28 percent) or gram negative bacilli (19 percent) organisms (2). Joint infections secondary to anaerobes are less likely and account for 2-3% of all cases (3). A review of the literature shows less than 50 documented cases of septic arthritis due to Clostridium species. Amongst these cases Clostridium perfringens is the most commonly isolated pathogen (4). To date there are no documented cases of joint infections secondary to Clostridium innocuum species.

Clostridium innocuum is a non-motile, anaerobic, gram positive organism that reproduces by sporulation. These organisms are normally found as a part of the usual human gut flora and are rarely human pathogens. The name “innocuum” is derived from the term “innocuous” to convey the innocence of these organisms as they do not produce clostridial exotoxins. A review of the literature shows fewer than 20 reported cases of Clostridium innocuum infections with most reported cases being described in immunocompromised patients such as those with diabetes mellitus, chronic hepatitis, acquired immune deficiency syndrome (AIDS), leukemia, and organ transplantation (5-6). Clinically patients can present with a spectrum of symptoms which include fever of unknown origin, diarrhea/constipation, and non-specific respiratory symptoms. In almost all cases bacteremia ensued. Most cases were associated with a traumatic penetrating injury with few reported cases due to hematogenous spread (5-6).

Laboratory identification of Clostridium innocuum can be challenging due to its variable gram staining morphology and atypical colony morphology on differing culture media. Most traditional phenotypic methods can only reliably identify these organisms to the genus level as a Clostridium species. However, using mass spectrometry (MALDI-TOF) these organisms can be identified to the species level. Rapid identification of Clostridium innocuum from the subset of Clostridium species is clinically important as these organisms are the only known Clostridium species with intrinsic resistance to vancomycin (7). Although they do not possess clostridial exotoxins, these organisms are thought to have a lipopolysaccharide-like virulence factor and have a mortality rate comparable to toxigenic Clostridium species (7). Due to resistance to vancomycin, metronidazole, piperacillin and ampicillin-sulbactam are the alternative recommended first-line treatment options.

For this patient, following the results of the gram smear the patient was started on IV vancomycin but due to an adverse allergic reaction was switched to intravenous pencillin G and oral ciprofloxacin. He was subsequently taken to the operating room for incision and drainage and left hip revision arthroplasty with cup exchange. Blood cultures were collected post-operatively and showed no growth, possibly due earlier antibiotic administration. Susceptibility studies from Mayo Laboratories showed pan susceptibility to penicillin, piperacillin-tazobactam, ertapenem, clindamycin, and metronidazole. The patient was subsequently switched to intravenous penicillin and continued to show clinical improvement during his remaining hospital course.

References

  1. Horowitz DL, Katzap E, Horowitz S, Barilla-labarca ML. Approach to septic arthritis. Am Fam Physician. 2011;84(6):653-60.
  2. Ryan MJ, Kavanagh R, Wall PG, Hazleman BL. Bacterial joint infections in England and Wales: analysis of bacterial isolates over a four year period. Br J Rheumatol. 1997;36(3):370-3.
  3. Shah NB, Tande AJ, Patel R, Berbari EF. Anaerobic prosthetic joint infection. Anaerobe. 2015;36:1-8.
  4. Gredlein CM, Silverman ML, Downey MS. Polymicrobial septic arthritis due to Clostridium species: case report and review. Clin Infect Dis. 2000;30(3):590-4.
  5. Leal J, Gregson DB, Ross T, Church DL, Laupland KB. Epidemiology of Clostridium species bacteremia in Calgary, Canada, 2000-2006. J Infect. 2008;57(3):198-203.
  6. Lee NY, Huang YT, Hsueh PR, Ko WC. Clostridium difficile bacteremia, Taiwan. Emerging Infect Dis. 2010;16(8):1204-10.
  7. Chia JH, Feng Y, Su LH, et al. Clostridium innocuum is a significant vancomycin-resistant pathogen for extraintestinal clostridial infection. Clin Microbiol Infect. 2017;23(8):560-566.

-Noman Javed, MD is a 3rd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

-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.

Hematopathology and Molecular Diagnostics Case Study: A 63 Year Old Man with Fatigue

The following case is an interesting overlap of Hematopathology and Molecular Diagnostics, and shows the utility of sequencing to detect a cancer before biopsy could.

A 63 year old gentleman presented to a heme/onc physician with six months of intractable anasarca, fatigue, and a recent mild thrombocytopenia (Table 1). They were otherwise in healthy condition. The physician initiated a lymphoma work-up that included a bone marrow biopsy. The tests were negative for M-protein.

Table 1. Summary of symptoms and relevant abnormal labs.

The bone marrow biopsy was somewhat limited, but the core contained multiple marrow elements. After a thorough review by a Hematopathologist, no evidence of dysplasia or other irregularities could be detected (Image 1). Flow cytometry detected no aberrant blast population. Cytogenetics detected 20del [16/20] and 5del [3/20]. These findings did not clearly indicate a specific diagnosis.

Image 1. 40x view of the bone marrow specimen at the initial presentation. No evidence of dysplasia was found.

As the clinical suspicion for a malignancy was high, the bone marrow specimen was sent for sequencing on a 1385-gene panel test. The test included tumor-normal matched DNA sequencing (“tumor” sample: bone marrow, normal: saliva), RNA whole transcriptome sequencing on the bone marrow, and Copy Number Variant (CNV) analysis. Tumor-normal matched sequencing helps rule out variants that are normal and present in the patient.

Somatic mutations were determined as those that were present in the “tumor” sample and not in the matched normal sample. The somatic variants found are listed below with their variant allele frequency (VAF) in parenthesis. Recall that a VAF of 40% means that a mutation is present in the heterozygous state in 80% of cells.

  • IDH2 (p.R140Q, 46%)
  • SRSF2 (p.P95T, 51%)
  • CBL (p.R499*, 47%)
  • KRAS (p.K117N, 12%)
Figure 1. View of IGV, which displays the NGS reads for IDH1 along with the variant allele highlighted in red. The color of the bars indicates the direction of the reads (forward in red and reverse in blue). This reflects the allele frequency of approximately 50%.

The mutations in these genes are commonly found in myeloid cancers including myselodysplastic syndrome. Activating mutation in IDH2 (isocitrate dehydrogenase 2) increase the production of the oncometabolite 2-HG, which alters methylation in cells taking them to an undiffereitiated state. SRSF2 (Serine And Arginine Rich Splicing Factor 2) is a part of the spliceosome complex, which regulates how sister chromatids separate from each other. Failures in the proper function of the complex creates genomic instability. CBL (Casitas B-lineage Lymphoma) is a negative regulator of multiple signaling pathways, and loss of function mutations (as seen here) lead to increased growth signals through several tyrosine kinase receptors. KRAS (Kirsten RAt Sarcoma virus) is an upstream mediator of the RAS pathway, which acquires mutations that lead to constitutive activation and sends growth signals to cells causing them to proliferate.

Furthermore the CNV analysis also found the heterozygous loss of chromosome 20 as reported in cytogenetics. CNV analysis did not detect chromosome 5 deletion, as it was below the limit of detection (20% for CNV analysis).

Figure 2. This plot shows the normalized read frequency of genes across each of the chromosomes is shown here. The drop at chromosome 20 is shown in a pale brown color on the right side of the graph. This is consistent with the cytogenetic findings. The loss of 5q isn’t seen as it is below the limit of detection of 30%.

These mutations are all individually common in MDS, but the co-occurance of each gives very strong evidence that MDS is the diagnosis (Figure 3). There have also been studies that provide prognostic implications for several of the genetic mutations present. Some mutations like SRSF2 or CBL at high VAF (>10%) indicate a poor prognosis, but mutations in IDH2 or TP53 at any frequency have not only a high chance of progression, but also a faster time to onset of disease. Another non-genetic risk factor for developing MDS is an elevated RDW, which we saw in our patient.

Figure 3. From Becker et al 2016.

All of these high-risk factors together led us to push for a diagnosis of MDS based off of molecular findings, and the patient was started on treatment with Azacitadine. Our assessment was confirmed 3 months later when, the patient’s follow up bone marrow biopsy showed significant progression with megakaryocytic and erythroid dysplasia and hyperplasia and reticulin fibrosis MF2 (Image 2). Aberrant blasts were detected (1-2%), but not elevated. This demonstrates how molecular findings predicted and predated the patient’s rapid progression to morphologic disease.

Image 2. Dysplastic, hyperplastic megakaryocytes and erythroid lineage.

In summary, multiple molecular mutations indicative of MDS were found in a symptomatic patient’s unremarkable bone marrow biopsy months before a rapid progression to MDS.

References

  1. Steensma DP, Bejar R, Jaiswal S et al. Blood 2015;126(1):9-16.
  2. Sellar RS, Jaiswal S, and Ebert BL. Predicting progression to AML. Nature Medicine 2018; 24:904-6.
  3. Abelson S, Collord G et al. Prediction of acute myeloid leukemia risk in healthy individuals. Nature 2018; 559:400-404.
  4. Desai P, Mencia-Trinchant N, Savenkov O et al. Nature Medicine 2018; 24:1015-23.
  5. Becker PM. Clonal Hematopoiesis: The Seeds of Leukemia or Innocuous Bystander? Blood.2016 13(1)

-Jeff SoRelle, MD is a Chief Resident of Pathology 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 improving genetic variant interpretation.

The Power of the Pause

The majority of laboratory injuries and exposures are preventable, and most of them occur because staff is not paying close attention to the situation. They lose their situational awareness or were never paying attention to it from the start. Unfortunately, lab safety professionals spend much of their time investigating such incidents rather than being able to prevent them. If laboratory staff could understand the power of the pause, labs would have fewer dangerous incidents.

One illustration of that power can be seen in a simple exercise. A group of people is asked to read aloud quickly a list of words that indicate different colors- green, red, etc. The words themselves, however, are written in different colors, and the colors do not match the words. For example, the word “red” is written in black, the word “blue” is written in green, etc. This first part goes well, you’re just asking them to read the actual words. Next, however, it gets harder. The people are asked to quickly go down the list again, but this time they are asked to say the color of the word, not that actual word. Typically, this does not go well. For the next step, the exercise is repeated at a much slower pace, with a slight pause between each word. Once a pause is placed between each word, the people recite the correct colors. The incongruent words and colors creates what is known as the “Stroop Effect,” first theorized in 1935, but pausing is a means of overcoming this issue in our brains.

When investigating a needle stick incident, the lab safety officer learned the employee completed the draw, attempted to engage the needle safety device, but stuck their finger when grabbing the needle to toss it into the sharps container. She did not notice the safety device did not engage and the needle was still exposed. The employee stated she was busy and in a hurry because there were many other patients waiting. I have always said that when a lab employee is stressed and busy, that’s when stopping for a moment to gain situational awareness is most important. Had this employee paused for a moment to ensure the needle safety device was fully engaged, the incident would never have occurred.

The lab manager had to speak to a chemistry tech after a serum splash exposure to the eyes. When looking at the work area, the manager noticed there was an adjustable face shield in place but that staff moved it into place only when needed. The tech admitted he was busy at the time of the splash and that he neglected to move the shield into place before uncapping specimens. Again, a pause to think about safety here would have helped.

In another situation, a microbiology technologist was eager to start the day and get it done since her vacation began the next day. She quickly went through the daily checklist and checked items off but did not actually perform the checks. Halfway through the day, she noticed it seemed warm and that it was unusually quiet at her biological safety cabinet work station. She decided to look at the gauges and noticed that there was no protective air flow in operation. She had been working with TB samples all morning. When she reported the issue, the manager told her that all employees in the area would need to go to Employee Health and be followed up for TB exposures. Pausing to perform the safety checks at the beginning of the shift would have made a big difference in that outcome for several employees.

Pausing for safety in the laboratory setting can be a powerful tool, even during the busiest moments. In fact, that’s when it works best. Use that pause in your arsenal, and teach maintaining situational awareness with your staff so that future injuries and exposures can be prevented.

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.