The Crown

Hello again everyone! Welcome back.

No, I’m not talking about Netflix or HRH Queen Elizabeth II, nor am I making references to tiaras, bars, beer brands, or imminently deliverable babies…I am, of course, talking about Coronavirus as it would certainly have caught most of our collective attention in the media by now.

I really enjoyed writing last month’s list of what I think are important things on the horizon for pathology and laboratory medicine this new year, but this month let’s take a more topical turn. So put your surgical masks on, wash your hands, quarantine the next 10 minutes of your time and get ready as I take a shot at the novel 2019 coronavirus outbreak!

***Let’s talk about you and me, Let’s talk about COVID-19…***

Less-Than-Common Cold

A long time ago, in a galaxy far, far away (aka: last year, about 20 minutes north of my apartment in Manhattan) I was in medical school, on rotations on the floors of a hospital in the Bronx. I experienced the surges of two flu-seasons and had a fantastic little mnemonic to remember the viruses that caused colds in most patients. Depending on age and immune system status, you had to think about the principal three viruses we see all the time—I remembered them as: “c-A-r,” note the capital “A.” Let me explain; the letters correspond to coronavirus, adenovirus, and rhinovirus. The are in a general order of when they appear during the months of the year (as coronavirus and rhinovirus kind of switch off in the spring, while adenovirus is around always thus is capital designation). There are a few hundred viruses which contribute to cold/flu-like symptoms in patients and, unless a patient is compromised in some way, we really worry most about one of them. Hint: it’s the one we give shots for annually, more on that in a minute.

Image 1. Okay this is a little better than my seasonal cold “cAr”…Seasonal variations in frequency of selected upper respiratory tract infection pathogens. Funny that, of all the colors, Influenza gets the good ‘ol fashioned “red alert” from the commanders on the USS Medscape. PIV = parainfluenza virus; RSV = respiratory syncytial virus; MPV = metapneumovirus; Group A Strep = group A streptococcus. (Source: Medscape)

As far as this coronavirus outbreak is concerned, this is a “novel” (i.e. new) variant (read: mutation) of a respiratory viral pathogen that is affecting a disproportionate number of patients in higher severity than expected. Its official entity name has now been filed by the World Health Organization (WHO) as COVID-19—corona virus disease of 2019. The actual virus is a relative of the infamous SARS virus from the early 2000s. That was SARS, this is SARS 2.0—literally. This virus is designated SARS-CoV-2. SARS stands for Severe Acute Respiratory Syndrome and is caused by strains of coronavirus found in the remnants of infected individuals’ coughs and sneezes—please wash your hands—and causes a spectrum of symptoms from mild to severe including pneumonia, respiratory disease, and even renal failure.

Image 2. Heeeerrreee’s COVID, well at least a coronavirus model. Named after the crown-like spikes on its envelope surface which act as receptor anchors in order to attach to cells and infect us. The receptors’ favorite target? ACE2 (angiotensin converting enzyme 2) receptors—am I getting your blood pressure up yet? I said renal failure…full circle, right? (Source: Centers for Disease Control and Prevention)

How Does this Even Happen?

Okay, who took a sabbatical to Wuhan, China, and ate a wild fruit-bat salad? No one, that’s not how this works. But, if you’re looking for quick grocery store recommendations at the present moment I’d probably tell you to check out ALDI or a farmer’s market a few spots higher on the list than the Huanan Seafood Wholesale Market in Wuhan which harbored a majority of outbreak case-cause tracings. The bottom line is that COVID-19 and the SARS-CoV-2 have appeared in the world the same way the previous similar outbreaks have—through zoonotic mutations which then spread to humans. This zoonotic transmission is so effective to presenting humans with super infectious entities because it sends us pathogenic material we would have never seen before and our “naïve” immune systems are caught off guard. Now don’t get all panicky; yes, I’ve seen Contagion, Outbreak, and read The Andromeda Strain—in fact, I absolutely love when epidemiological medicine has the media spotlight. It’s a very exciting way to showcase public health, medicine, and—our favorite—laboratory professional work!

Image 3. Okay, no bat salad but this outbreak does involve bats endemic to the regions in question. It also involves, pangolins, kind of like armadillos, which are bought and sold (and trafficked) all around the world for various reasons. The pangolin DNA matches the SARS-CoV-2 genome highly, so scientists assert that its involvement is inherent. The complications in sanitation and animal/food handling are obviously implicated in this and other outbreaks, but dealing with COVID-19 requires a strong public health response. (Source: Osmosis)

Basically, this process of mutation and transmission is the modus operendi of a viral particle. You can’t quite kill them, they’re not quite alive by biological definitions, they’re just packaged proteins on autopilot. They’re kind of like natural robots that want to propagate their species by adapting over time—they’re The Borg or Cybermen, depending on your sci-fi preferences. But both offending automaton predators have a mutual enemy in public health—a doctor (get it? TARDIS pilot and/or Beverly Crusher both work wonders in a pinch…) Anyway, it’s never just physicians, but a whole hard-working team of health advocates that conduct surveillance, field research, epidemiologic studies, and first-hand treatment.

***Side note: if you’re bored, in a hurry, or just don’t like my articles—don’t fret! Go watch that Osmosis video on COVID-19 and you’ll be up to snuff on the current outbreak in no time. Or in 12ish minutes.***

***Hey, you made it this far. Great! Interested to know more about the COVID-19 virus from our very own American Journal of Clinical Pathology? Visit here to learn more about the story of how this pesky coronavirus mutated its way into headlines. Fresh off the AJCP presses this month!***

You Should Update Your Antivirus Software

Image 4. Fake news is the new normal. It’s hard to sift through a sea of garbage. You can’t just ignore it, because sometimes there’s serious situations like war—or viral pandemics! So, you’ve got to update your personal mental anti-spam filter and know how to sniff out suspicious sources. Let’s call it “information hygiene” to be consistent with the zoonotic exposure narrative, yea? (Image source: NPR)
Image 5. Let’s lighten the mood a bit. These situations can drive folks absolutely nuts. People obsess over minutiae and often wrong details when archetypal preventions like hand-washing and isolation are paramount to prevent epidemics. Public health and pandemic situations breed conspiracy thinking—wait, never mind, this is a photo of me explaining the residency match process to my friends and family. Kidding. Kind of. (Source: a popular meme, but originally from It’s Always Sunny in Philadelphia)

No doubt in my mind you’ve probably seen plenty of coverage about SARS-CoV-2 in the media. I’d also be willing to bet a lot of it is either dilute, sensational, or possibly even misleading. Regardless, there are always going to be people that don’t “buy in” to the public health message. If you remember Contagion¸ Jude Law’s character pushes the efficacy of “forsythia,” a homeopathic herb supplement that supposedly mitigates the horrible disease spread from southeastern Asia from improper food handling—if I recall correctly, it was a paramyxovirus that time. In this SARS-CoV-2 epidemic we have no current effective treatments, so prevention is key.

In an effort to address this type of health misinformation the WHO and CDC are actively disseminating as much educational information and graphics as they can write. Trying to dispense advice for the public including proper mask wearing, education videos, and myth-busting (i.e. hand dryers do NOT kill the COVID-19 virus, UV lamps do NOT kill the virus, thermal readers are effective in screening populations for symptoms within limitations, alcohol and chlorine do NOT kill the virus, receiving packages from China is still safe, pets don’t harbor the virus at this time, other vaccines do not affect this virus, saline nose sprays do not affect this virus, garlic/oils/other supplements have no effect on this virus, and all age groups are affected)—good stuff there. The most trusted sources of information regarding epidemics should be the representatives of functional medicine and health outcomes, doing work every day to make people healthier. Often times, politics, misinformation, or complex situations make information delivery harder than you’d think and the risks are increasingly high.

Image 6. BONUS MATERIAL UNLOCKED: Glad you made it this far. I mentioned Netflix’s The Crown above, but if you’re looking for a fantastic series that breaks down public health epidemiology, influenza burden, research and surveillance on emerging pathogens as updated as 2019, check out Pandemic. 5/5 surgical masks would binge again. (Source: Netflix)

A Crown of Thorns: Don’t Forget About the FLU!

Flu vaccine deniers: turn away now or be healed! —or at least exposed to another point of view rooted in evidence-only concepts in medicine and population health. Consider the following: as of this month, COVID-19 has infected 43,000 people and killed 1,000 (approximately 2-3%). Remember SARS? That infected 8,000 and killed 700 (approximately 10%). MERS? 2,500 infected, 860 deaths (approximately 34%). And what about Ebola? 29,000 infectious cases with 11,000 deaths (approximately 40%). That was sourced from the Osmosis video with data from the WHO. Pretty impressive right? Well, not if you look at this: according to the CDC, the 2019-2020 influenza burden statistics include 36,000,000 infectious cases, with 17,000,000 clinical visits, 440,000 hospitalizations, and 36,000 deaths. One might say “hey, Dr. Kanakis, slow down there you’re spitting out all these numbers and the facts won’t lie. Looks like influenza only killed 0.1% of cases.” And you know what, you’re right. 0.1% is lower than the other viral epidemics. But check this out, because of the sheer number of cases, that means more people died of influenza than COVID-19, SARS, MERS, and Ebola COMBINED and those happened in other years. That‘s just this year’s flu season alone. I’ve talked before about recognizing and detecting the common cold vs. influenza before, check it out if you’d like a refresher!

Image 7. What’s a statistical percentage data point if it’s a relative, a loved one, or yourself? Protect yourself, protect others, and let’s find ways to make proper health knowledge available and acceptable to the masses while promoting excellent healthy behaviors, together! (Image source: New York Post)

We have influenza every single year, and it kills so many more people than we realize. If you want to talk about a terrifying, global viral epidemic, we’ve already got one. And it’s closer than you think. So wash your hands, reduce exposures if you’re sick or immunocompromised, get proper rest, eat well, exercise, read my articles every month, but most importantly—and I cannot stress this enough—get your FLU SHOT!

Thank you so much, see you next time!

Constantine E. Kanakis MD, MSc, MLS (ASCP)CM completed his BS at Loyola University Chicago and his MS at Rush University. He writes about experiences through medical school through the lens of a medical lab scientist with interests in hematopathology, molecular, bioethics, transfusion medicine, and graphic medicine. He is currently a 2020 AP/CP Residency Applicant and actively involved in public health and education, advocating for visibility and advancement of pathology and lab medicine. Follow him on Twitter @CEKanakisMD

Microbiology Case Study: A Teenager with an Infected Wound

Case History

A male teenager presented to the emergency department following a 4 wheeler accident. He sustained extensive trauma to his right lower leg with a large, dirty laceration and grossly exposed muscle. His pulses were intact and motor & sensory nerve function were preserved. The wound was irrigated at the bedside and the patient was admitted with a plastic surgery consult for wound coverage. Cefepime was empirically started. After 10 days in the hospital and multiple surgeries to care for the wound, the patient developed a fever and increased pain, erythema, and swelling at surrounding the wound. The trauma service ordered blood and wound cultures.

Laboratory Identification

Image 1. The wound culture grew lavender-green colonies on blood agar (48 hours of incubation in CO2).
Image 2. Clear, lactose-negative colonies on MacConkey agar (48 hours of incubation in CO2).
Image 3. Gram stain from the blood agar plate showed gram negative rods (100x, oil immersion).

The oxidase reaction was negative. MALDI-TOF mass spectrometry identified the isolate as Stenotrophomonas maltophilia from the wound culture. Blood cultures were negative.

Discussion

Stenotrophomonas maltophilia is a common non-fermenting gram negative rod that is ubiquitous in moist environments but is not commonly a member of human flora. S. maltophilia can readily be isolated from hospital surfaces and those with traumatic injuries, prolonged hospitalizations, on mechanical ventilation, and with in-dwelling devices are more susceptible to nosocomial infections by this organism. Those who are immunocompromised or have cystic fibrosis are also at an increased risk.  

In the laboratory, S. maltophilia is characterized as an aerobic, Gram-negative rod that grows as lavender-green colonies on blood agar (Image 1) and has an ammonia-like odor. This organism is catalase and oxidase negative and DNase positive. S. maltophilia is motile and is able to utilize glucose and maltose by oxidative fermentation. Current automated identification systems and MALDI-TOF mass spectrometry are able to accurately identify S. maltophilia.  

S. maltophilia is intrinsically resistant to many broad-spectrum antibiotics, including carbapenems and aminoglycosides. Beta-lactam resistance is due to two beta-lactamases and renders beta-lactam inhibitors ineffective. Trimethoprim-sulfamethoxazole (TMP-SMX) is the antibiotic of choice to treat S. maltophilia infections; however, resistance can develop. In the case TMP-SMX resistance, ceftazidime, minocycline, ticarcillin-clavulanate, ciprofloxacin, and levofloxacin can be tested. 

In the case of our patient, susceptibility testing was performed on the Vitek2 instrument and the isolate was susceptible to TMP-SMX. He was switched to TMP-SMX and underwent additional surgical procedures to wash out the infected area.

-Karla Perrizo, MD, is a Clinical Pathology resident at the University of Mississippi Medical Center.

-Lisa Stempak, MD is the System Director of Clinical Pathology at University Hospitals Cleveland Medical Center in Cleveland, Ohio. She is certified by the American Board of Pathology in Anatomic and Clinical Pathology as well as Medical Microbiology. Her interests include infectious disease histology, process and quality improvement, and resident education.

Biomarker Testing for Cancer Patients: Barriers and Solutions Part 3

This month we will continue discussing the common barriers to biomarker testing for cancer patients in the community.

As you may recall, these are the top 10 barriers that I’ve seen to biomarker testing in the community:

  1. High cost of testing.
  2. Long turnaround time for results.
  3. Limited tissue quantity.
  4. Preanalytical issues with tissue.
  5. Low biomarker testing rates.
  6. Lack of standardization in biomarker testing.
  7. Siloed disciplines.
  8. Low reimbursement.
  9. Lengthy complex reports.
  10. Lack of education on guidelines.

As I mentioned last month sample quantity and quality are both important when considering biomarker testing. We covered tissue quantity issue last month; let’s move on to tissue quality issues. I will focus on what happens to the tissue prior to testing being performed, called the preanalytical phase of testing. We now know that preanalytical variables can alter protein structure, DNA, and RNA (1). This can alter the results that are used to diagnose and treat patients. Despite the impact to downstream assays, this area is typically poorly controlled. Some of the sources of variance due to preanalytical processes include procurement, fixation, processing, and specimen storage (1).

Procurement:

ROSE: I covered rapid onsite evaluation (ROSE) during procurement last month with regards to tissue quantity. Having a pathologist evaluate both tissue quality and sufficiency during the biopsy is invaluable. For molecular analysis, areas of necrosis should be avoided.

Fixation:

Time to get specimen into fixation (cold ischemic time): Tissue begins to degrade as soon as it is removed from the body. The amount of time between removing tissue from the body and fixing it is referred to as the cold ischemic time. This time should be as short as possible. Less than 1 hour is recommended if molecular studies are going to be performed on the specimen (1). Tissue begins degrading immediately and this degradation can affect the results of biomarker test such as IHC & FISH (1). I’ve seen cases where the biopsy was collected, but due to a busy day in the OR, it was not sent to pathology immediately. This can cause the tissue to be so degraded that testing cannot be performed. Unfortunately, sometimes we don’t know there was a fixation issue until the molecular assay fails repeatedly. This is an expensive way to determine something went wrong in the preanalytical phase.

For small biopsies such as core needle biopsies or even fine needle aspirates, one way to decrease time to get the specimen into fixative is by having the fixative in the suite where the biopsy is collected so that it is put into fixative immediately. This won’t work for large pieces of tissue that may need to be dissected for optimal fixation penetration. For these biopsies, diligence needs to be taken to get the biopsy to the laboratory as soon as possible. Multidisciplinary communication will help ensure the hand-off occurs in a timely manner. Specimens may need to be dissected for optimal fixation penetration.  Other specimens may need to be decalcified prior to fixation.  

Decalcification: Most decalcification processes with formic acid have negative downstream effects on molecular testing. We have about a 50% success rate of PCR actually being able to amplify DNA after standard decal due to degradation. The solution to this is to use EDTA-based decalcification; however this could lengthen the fixation time drastically. Some new gentle decals are on the market that could be used without increasing the turnaround time.

Proper Fixative: We also need to ensure specimens are fixed in the appropriate fixative. The most common and widely accepted fixative is 10% neutral buffered formalin (NBF).  DNA yield and some downstream biomarker tests are negatively impacted if unbuffered formalin is used (1). Tissue fixation in alcohol such as 70% ethanol may be even better than NBF if the downstream assay involves DNA extraction; however ethanol fixation may negatively impact IHC or FISH assays (2).

Time in Fixative: Studies show that the appropriate amount of time a biopsy needs to be in fixative is about 6-72 hours (1). The wide range of times is due to the range of specimen sizes, it is generally accepted that formalin penetrates tissue at the rate of 1 mm/hour (3). Tissue may need to be dissected to ensure complete fixation within a reasonable time. If the tissue is not fixed appropriately, the tissue will degrade. However, too much time in fixative can lead to degraded DNA.

Downstream biomarker assays such as FISH, PCR, and ISH can be affected if the fixative time is greater than 72 hours (1). Small community sites that do not have someone processing specimens over the weekend may need to adjust their biopsy schedules on Fridays to ensure specimens do not sit in fixative for more than 72 hours. Unfortunately, unless the specimen is a breast biopsy, in which CAP requires fixation times to be documented and controlled, fixation times are rarely documented and controlled. This is an area we could all improve on. It would be nice to know how long a specimen was stored when we are troubleshooting a downstream assay.

Processing:

The impact of processing variables on biomarker testing has not been well published. One variable that has been called out as having a negative effect on PCR is mixing beeswax with paraffin wax. Only pure paraffin wax should be used. Other changes to the processing schedules should be performed with coordination between departments. If the anatomic pathology laboratory makes changes to their process, a verification study on the impact to the downstream assays should be performed. How extensive that study is should be determined by the medical director.

Specimen Storage:

As long as the specimen was properly fixed, FFPE block storage is normally fairly hearty. The literature recommends blocks that are used for molecular analyses be less than 10 years old (1). Although I wouldn’t recommend using blocks older than 10 years, cases positive for some biomarkers are rare, so during our validations some blocks used were over 10 years old, and the downstream PCR-based assays still worked. However, I have no way to know if the DNA yield was compromised.

Unlike FFPE blocks, long-term storage of FFPE slides does affect downstream testing (4). Room-temperature storage seems to be worse than refrigerated storage of slides. Slides should be used for IHC and biomarker testing relatively quickly (< 30 days).

Unfortunately I cannot cover all sources of preanalytical error thoroughly in a 1,000 word blog post, but hopefully this sparks your interest enough to check out the references where the authors give a more detailed explanation of preanalytical issues.

References

  1. Bass BP, Engel KB, Greytak SR, Moore HM. A review of preanalytical factors affecting molecular, protein, and morphological analysis of formalin-fixed, paraffin-embedded (FFPE) tissue: how well do you know your FFPE specimen? Arch Pathol Lab Med. 2014;138:1520-30.
  2. Lindeman NI, Cagle PT, Aisner DL, Arcila ME, Beasley MB, Bernicker EH, et al. Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. Arch Pathol Lab Med. 2018;142:321-46.
  3. Howat WJ, Wilson BA. Tissue fixation and the effect of molecular fixatives on downstream staining procedures. Methods. 2014;70:12-9.
  4. Economou M, Schoni L, Hammer C, Galvan JA, Mueller DE, Zlobec I. Proper paraffin slide storage is crucial for translational research projects involving immunohistochemistry stains. Clin Transl Med. 2014;3:4.   

-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. 

A Trainee Abroad: One Cytopathology Fellow’s Experience at a Teaching Hospital in Rwanda

The University Teaching Hospital of Kigali (CHUK) is the largest hospital in its District of Nyarugenge and the biggest national referral hospital in the country of Rwanda, with a 565 hospital bed capacity and 6 operating theaters. It is located in the heart of the capital of the country, Kigali, contributing to its easy accessibility by patients. Rwanda is a country of over 12.5 million people, with an estimated 70.2% of the population living in a rural setting. Per the World Bank, there is an estimated 1 physician per 10,000 people in-country. The government of Rwanda is focused on elevating the country from a low-income developing nation to a middle-income country with a robust health sector capable of ensuring a healthy people with adequate healthcare access. It provides universal healthcare, at a small cost, to all Rwandan citizens who aren’t provided health insurance through employment. In Rwanda there are a total of 14 practicing pathologists, which equates to approximately 1.1 pathologists per million people in the country. In contrast, within the United States there are an estimated 60 pathologists per million people. CHUK offers an array of outpatient, inpatient, surgical, and diagnostic medical services. Inpatient and outpatient services include surgery, accident & emergency, internal medicine, mental health, anesthesiology & critical care, gynecology, pediatrics, maternal & neonatology, ear/nose/throat, ophthalmology, neurosurgery, pediatric surgery, urology, nephrology, dialysis, oncology, and dermatology. Surgical services include general surgery, general pediatric surgery, neurosurgery, orthopaedics, ophthalmology, ear/nose/throat, and obstetrics/gynecology. Diagnostic services include ultrasound, digital x-ray, CT scan, and anatomic and clinical pathology services. In its current state, the hospital has a total of 18 divisions.

There are two facets to the pathology laboratory at CHUK: the Anatomic Pathology (AP) and the Clinical Pathology (CP) laboratories. Within the AP laboratory, also known as the histopathology laboratory, all surgical specimens are grossly examined by a pathology resident and/or pathologist, prepared by a pathology resident for processing, and processed by laboratory technicians into formalin-fixed paraffin-embedded tissue placed onto glass slides. These glass slides are then reviewed by both the pathology residents and the pathologists in order to render a diagnosis, which is communicated to the clinician in order to help direct appropriate patient management. Specimens reviewed at CHUK are predominantly “in-house” specimens generated by the surgeons and clinicians functioning within the walls of the institution. “Referral” specimens are a rarity and generally consist of small biopsies. Cytopathology specimens are also processed within the AP laboratory and include a mixture of fine needle aspiration (FNA) specimens, obtained by pathology residents via superficial FNA, as well as exfoliative cytology specimens such as effusions and urines collected by “in-house” clinicians. Cervical screening conventional pap smears are a rarity. Within the AP laboratory, Diff-Quik, Papanicolaou, and hematoxylin & eosin (H&E) staining was available for slides, as well as a limited panel of special stains: PAS-D, auramine, and a modified acid-fast stain. No immunohistochemistry was available on-site, though cases could be sent for free to nearby Butaro Hospital for IHC or consultation via digital slide scanning.

Regarding my experience at CHUK, I departed the United States on a Saturday evening and reached Kigali, Rwanda by 1AM the following Monday morning. On my first day at CHUK, I was introduced to the 5 anatomic pathology staff, 9 anatomic pathology residents, and the single visiting pathologist serving as a laboratory inspector conducting a mock inspection/assessment. I was given a tour of the pathology facilities as well as the entire hospital system.

There were two aspects to my primary job at CHUK: teaching the residents cytopathology and microscopic review of all live cytopathology cases received in the laboratory. Regarding resident education, there were four ways in which I interacted with the residents during my time to facilitate cytopathology education: lectures, multi-headed microscope unknown slide sessions (unknown case conference where I provided the residents with cases they had never seen before), multi-headed microscope “stump the chump” unknown slide sessions (where the residents presented me with unknown cases I had never seen before), and interactive practicals where we performed various hands-on aspects of cytopathology and general pathology practice.

In respect to lectures, I delivered a total of eight 1.5 hour powerpoint-based lectures covering the following topics: breast cytology, thyroid cytology, lymph node cytology, salivary gland cytology, urine cytology, effusion cytology, peritoneal washing cytology, and frozen section pathology (frozen section lecture presented as a combined effort with Dr. Raina Flores). For unknown slide sessions in which I presented cases to the residents, we had 6 sessions covering the following topics: breast, thyroid, salivary gland, urine, conventional pap, and cerebrospinal fluid. We completed a total of 5 “stump the chump” sessions, where residents gave me slides that I had never seen before and we discussed each case and its work-up as well as its associated differential diagnosis or final pathologic diagnosis at the multi-headed microscope. Topics covered included: breast, thyroid, salivary gland, lymph node, and effusions. Finally, with the assistance of “in-house” pathologists, I helped conduct 2 hands-on practicals with the residents: the first regarding fine needle aspiration technique and slide smearing technique (with Dr. Claire Nadyisaba) and the second regarding performance of frozen section intraoperative consultations using Leica CM1850 cryostats and cow liver (with Dr. Raina Flores).

The second of my duties, live cytopathology case review, was also performed at the multi-headed microscope with the residents each afternoon. On a given day, we would typically receive somewhere between 1 and 4 FNA consultations for which the residents would go to FNA clinic and perform the procedure. The laboratory also received various aspirated and exfoliative cytology specimens, such as pleural effusion and ascites fluids, from clinicians within the hospital system. In total, we reviewed 51 cytopathology cases together at the microscope. 27.5% were neoplastic, with 7.8% being malignant and 2% being lymphoma. 56.8% of cases were negative for malignancy, with 21.5% being inflammatory/infectious. In total, 9.8% of cases were interpreted as “atypical” and 5.9% of cases were non-diagnostic. Of the 51 cases, 21 (41.2%) were FNA consultations that I attended and the resident performed.

On my final day of work, I provided the residents with a 41-page cytology knowledge assessment (in PDF format) to complete at their leisure. This test covered the following topics: cervical and vaginal cytology (19 questions), urine and bladder cytology (11 questions), effusion cytology and peritoneal washings (13 questions), cerebrospinal fluid cytology (12 questions), breast cytology (8 questions), thyroid cytology (17 questions), salivary gland cytology (13 questions), and lymph node cytology (11 questions). Within the document, an answer key with associated detailed explanations was provided so it could serve as a learning aid/study guide for the trainees.  On my last workday, the residents were asked to evaluate their experience with the Cytopathology Module/Course. A total of 7 of 9 residents completed the evaluation. Regarding preparation and organization of different topics, all residents found the quality of the powerpoints to be “very good” or “excellent”. The quality of the practical sessions was rated as “good,” “very good” or “excellent by all residents and the entire module was given an overall rating of “very good” or “excellent” by all of the residents. The majority of residents felt their time was used effectively during this module and that the venues for theoretical and practical learning were appropriate. In the free-text areas for additional comments, suggestions for improvement included a longer duration (at least 4 weeks) of the module, more hands-on practical time, the opportunity for residents to present information, and more microscopy sessions. For additional topics to be covered, respiratory cytology was suggested. In overarching comments regarding their module experience, the residents felt the module was well-prepared, the teaching sessions were well-organized, and that the course was interesting and helpful.

Finally, though not within the confines of my assigned “duties”, I also spent a portion of each day acting as “consultant” to the on-site pathologists for challenging surgical pathology cases, offering opinions as able for various lesions that were challenging to classify on H&E morphology alone. I also served as a “second reviewer” for new malignant diagnoses being rendered in the laboratory, offering my name to be included in the report as a board certified pathologist who has laid eyes on the case and agrees with the interpretation. Examples of some interesting surgical pathology cases I saw in “consultation” included Wilms tumor (nephroblastoma), cystic partially differentiated nephroblastoma (CPDN), pleomorphic xanthoastrocytoma (PXA), sinonasal undifferentiated carcinoma, basaloid moderately-differentiated carcinoma of the uterine cervix, high-grade large cell lymphoma of the cervical lymph node, high-grade squamous intraepithelial lesion of the vulva arising within a condyloma acuminatum, and low-grade papillary urothelial carcinoma of the bladder. I also attend a single Tumor Board Multidisciplinary Conference with two residents and 1 staff pathologist in which a resident presented a case of mucinous moderately-differentiated adenocarcinoma of the colon transmurally invading adjacent ileum. It was interesting to hear the clinicians, pathologists, and radiologists interact in addressing quality of care, efficiency of care, and clinical decision-making. The time of initial presentation to the time of surgery was greater than 1 year for this patient.

My time spent at CHUK in Kigali, Rwanda was an invaluable experience. The work setting granted me the opportunity to expand my role as an academic educator. I was offered the opportunity to present as many lectures as possible to the resident trainees, participate as the leader of multi-headed microscope slide sessions, serve as a spearheading physician in laboratory services expansion efforts, and work as an ‘attending’ physician overseeing trainees’ performance of FNAs. It was an experience that demanded personal growth, via the assumption of roles that I am not privy to as a post-graduate medical education trainee in the United States. Additionally, I was exposed to a cytopathology and surgical pathology workload for a patient population quite dissimilar from the community I am used to serving. With limited ancillary testing capabilities, I returned to a more “pure” form of rendering pathologic diagnoses, based on H&E morphology alone rather than on the synthesis of cyto- and/or histomorphologic appearance coupled with various ancillary diagnostic testing data points. In conclusion, this was an experience that expanded my understanding of the ways in which I can be useful as a board certified anatomic and clinical pathologist interested in incorporating medical mission work into my clinical practice. Beyond arriving in countries without expansive pathology laboratory systems and simply doing the work, I can also pursue opportunities where I can help educate and shape burgeoning in-country pathologists who will then go on to have productive, hopefully decades-long careers in their country, serving their countrymen. This trip certainly expanded my understanding of the role of a “visiting” pathologist. This experience was made possible by the ASCP Trainee Global Health Fellowship Award. Thank you so much to the ASCP, Dr. Dan Milner, Alpa Pandya, and the CHUK pathology department for helping to facilitate this opportunity!

Image 1. Dinner with CHUK pathologists and pathology residents
Image 2. Frozen section training with CHUK pathology residents
Image 3. CHUK laboratory medicine building
Image 4. CHUK hospital
Image 5. CHUK hospital entrance
Image 6. Small “downtown” area near CHUK hosptial–Kwibuka (“to remember”) memorial in remembrance of the 25th anniversary of the Rwandan genocide.
Image 7. Overlooking Kigali.
Image 8. Ferry ride to various neighborhoods in Kigali

-Kelsey McHugh, MD is a board certified anatomic and clinical pathologist, with cytopathology subspecialty certification, who is currently completing gastrointestinal, hepatic, and pancreatobiliary pathology subspecialty training. She anticipates graduating from the Cleveland Clinic Gastrointestinal, Hepatic, and Pancreatobiliary Pathology Fellowship in June 2020, after which she will remain at the Cleveland Clinic as a staff pathologist beginning July 2020.

Hematology Case Study: Thrombocytopenia in a 50 Year Old Male

A 50 year old male patient receiving chemotherapy for treatment of gastric cancer presented to ER. Labs reported: 

WBC = 5.4 x 103/μL

Hgb = 8.9 g/dL

PLT (impedance) = 26 x 103/μL

PLT-F (fluorescent) = 9 x 103/μL

IPF = 21%

The hemoglobin was consistent with the patient history. Flags on the original impedance platelet count included thrombocytopenia, platelet clumps and platelet abnormal distribution. The sample was checked for clots, with no clots found. A fluorescent platelet count (PLT-F) was reflexed and the critical platelet count was called to the ER physician. The high immature platelet fraction (IPF%) indicates increased platelet production. Despite the increased production, the patient still had a severe thrombocytopenia. This would suggest thrombocytopenia caused by platelet destruction or consumption. Examination of the blood smear showed the presence of moderate numbers of schistocytes.

Image 1. Schistocytes seen on peripheral blood smear

Additional labs were ordered. BUN and Creatinine were slightly elevated. PTINR and APTT were within normal range. LDH was markedly increased. The physician was able to use this information, along with the clinical presentation and history, to diagnose Thrombotic Thrombocytopenic Purpura (TTP). Plasma exchanges were initiated. The patient expired 3 days later.

The difference between the impedance platelet count and the fluorescent platelet count in this patient is actually related to the presence of schistocytes. With thrombocytopenia, platelet counts can be less reliable than with normal counts. Automated platelet counts were originally performed by impedance methods, then better accuracy and precision was obtained with optical platelet counts. Physicians rely on precision with very low platelet counts to make informed decisions about treatment. The problem with the impedance counts at the low end is that RBC fragments, schistocytes and microcytic RBCs can be counted as platelets, giving a falsely high count, as we see in this case. On the other hand, measuring platelets by size (optical) can miss large platelets leading to a falsely low count. The PLT-F is more reliable because it uses a platelet specific dye which eliminates these interferences. The fluorescent dye labels the RNA. Forward scatter is used to determine size while fluorescence is used to measure RNA content. With gating set based on cell volume and RNA content, the PLT-F can be measured. When there is an abnormal scattergram or a low platelet count, the PLT-F is reflexed and the IPF% is also reported.

The Immature platelet fraction (IPF) can also be used to help understand the etiology and aid in diagnosis. Historically, the MPV has been used as an indirect marker for platelet production. However, an inherent problem with the MPV is that, similarly to the impedance platelet count, this count can be unreliable because any RBC fragments or particles may interfere with the measurement. Reticulated or immature platelets are the youngest platelets, within 24 hours of being released from the bone marrow. Measurement of these is a concept that first emerged in the late 1960s, before automated hematology analyzers performed platelet counts. Thus, the original method was staining with new methylene blue and manually counting, much like a manual reticulocyte count. These manual methods tend to be tedious and imprecise. In the last 20 yeas we have developed flow cytometry methods for performing a reticulated platelet count. Reticulocytes are stained with Thiazole Orange and passed through a flow cytometer. Unfortunately, there is no standardization for the procedure as there are variations in dye concertation, timing and gate settings. As well, this method is also time consuming, labor intensive, costly, and requires highly trained technologists to perform.

Newer flow cytometry methods to count these youngest platelets are available on Sysmex and Abbott CELL-DYN analyzers. The IPF (Sysmex) or RetPLT(Abbott) can be performed along with the routine CBC with no additional sample or time required. Knowing the reticulated or immature platelet fraction can help physicians to differentiate pathogenesis. A decreased percent of newly formed platelets may indicate that thrombocytopenia is caused by deficient platelet production, as seen in bone marrow failure. Increased circulating immature platelets with a low platelet count may suggest that the bone marrow is making adequate platelets and the thrombocytopenia is caused by platelet destruction or consumption. Treatment for these scenarios is different, and the physician must determine the etiology in order to determine treatment

Thrombotic thrombocytopenic purpura (TTP) is a microangiopathic hemolytic anemia with thrombocytopenia and organ failure caused by microvascular thrombosis. Platelets clump in the small blood vessels and cause the low platelet count. The hemolytic anemia causes schistocytes which can be seen on the peripheral blood smear. In this case, the low platelet count and high IPF, schistocytes on the smear and the patient presentation were all important factors that led to a speedy diagnosis and start of therapy.

Plasma exchange is the treatment of choice for TTP. With the advent of therapeutic plasma exchange, mortality from TTP has decreased from about 90% to 10-20%. In patients who have relapses or become refractory, vincristine has been used successfully as an adjunct to plasma exchange.4 The exact etiology of TTP is unknown. It can be secondary TTP, often triggered by chemotherapy drugs, or can be sporadic. Sporadic, or idiopathic, TTP is now thought to be associated with an acquired autoimmune deficiency of a plasma metalloprotease named ADAMTS13. The ADAMTS13 gene controls this enzyme, which is involved in blood clotting. In acquired TTP, the ADAMTS13 gene isn’t faulty. Instead, the body makes antibodies that block the activity of the ADAMTS13 enzyme. In these cases, a lack of activity in the ADAMTS13 leads to TTP. Almost all cases of recurrent TTP have severe ADAMTS13 deficiency. These patients benefit from immunosuppressive therapy with vincristine along with plasma exchange.

However, despite the decreased mortality seen with plasma exchange, patients with cancer, infections, transplant patients, or those receiving certain drug therapy have a much worse prognosis.4 In this case study, this was this patient’s first episode of TTP and he was undergoing chemotherapy for gastric cancer. The patient’s unfortunate outcome is most likely linked to this finding.

References

  1. Arshi Naz et al. Importance of Immature platelet Fraction as a predictor of immune thrombocytopenic purpura. Pak J Med Sci 2016 Vol 32 No 3:575-579
  2. Johannes J. M. L. Hoffmann, Nicole M. A. van den Broek, and Joyce Curvers (2013) Reference Intervals of Reticulated Platelets and Other Platelet Parameters and Their Associations. Archives of Pathology & Laboratory Medicine: November 2013, Vol. 137, No. 11, pp. 1635-1640.
  3. M Meintker, Lisa & Haimerl, Maria & Ringwald, Juergen & Krause, Stefan. (2013). Measurement of immature platelets with Abbott CD-Sapphire and Sysmex XE-5000 in haematology and oncology patients.
  4. J. Evan Sadler, Joel L. Moake, Toshiyuki Miyata, James N. George Clinical chemistry and laboratory medicine : CCLM / FESCC. 51. 1-7. 10.1515/cclm-2013-0252.; Recent Advances in Thrombotic Thrombocytopenic Purpura. Hematology Am Soc Hematol Educ Program 2004; 2004 (1): 407–423. doi: https://doi.org/10.1182/asheducation-2004.1.407
  5. Sysmex White Paper. The role of the Immature Platelet Fraction(IPF) in the differential diagnosis of thrombocytopenia. www.sysmex.com/us

-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.

Microbiology Case Study: A 60 Year Old with Non-Healing Wound

Clinical history

A 60 year old patient with a past medical history of type II diabetes mellitus, right Charcot foot, and cirrhosis presented to the emergency department with altered mental status and several days of subjective fevers, as well as a 2 month history of right lateral malleolar non-healing ulcer which had subacutely increased in size and volume of drainage.

The patient’s spouse reported the wound had been showing purulent discharge for 3 weeks. Nine days before presentation, the patient had seen a foot and ankle specialist for evaluation of his Charcot foot and the ulcer. Radiographs were taken at this time, but no treatment was initiated. Worsening of the wound was associated with an episode of long travel, after which the patient reported being able to see bone.

Infectious disease noted that the patient had a pet corgi.

On exam, the patient was febrile with dry mucous membranes and oriented only to person. MRI showed evidence of possible osteomyelitis. The patient subsequently underwent a right below the knee amputation.

Laboratory findings

Gram smear of a sample taken from the patient’s ankle wound in the emergency department showed many neutrophils, moderate gram positive cocci and moderate gram negative bacilli, with intraleukocytic organisms seen. Growth was observed on blood and chocolate agar plates, but there was no growth on the MacConkey plate. The organisms were identified as few Pasteurella multocida, few vancomycin resistant Enterococcus faecalis, and few usual skin flora.

Image 1. Gram stain of the sample taken from the patient’s ankle wound.

Blood cultures drawn in the emergency department were positive at 10 hours in both bottles, and again on planting showed growth on blood and chocolate agar, but no growth on MacConkey. The organism was identified as P. multocida, consistent with that which grew from the ankle wound culture.

The patient underwent a right below the knee amputation, and anaerobic cultures taken from the right foot again grew P. multocida.

Discussion

Pasteurella multocida is a nonmotile gram negative bacillus which is part of the normal oropharyngeal flora in domestic dogs and cats. It is a facultative anaerobe, positive for oxidase, catalase, and indole. It grows on chocolate and blood agar, forming small, gray, non-hemolytic colonies. It does not typically grow on MacConkey agar.

P. multocida is classically associated with a zoonotic soft tissue infection in humans who suffer bite wounds from a pet, as well as licking of any broken skin by a pet. These infections have a characteristic rapid onset and intense inflammatory response, and can progress to necrotizing fasciitis. Cases of Pasteurella osteomyelitis can be associated with significant wound infections. Conditions such as diabetes, liver dysfunction, and organ transplantation can predispose patients to Pasteurella bacteremia.

Pasteurella spp. are susceptible to beta-lactam antibiotics in most cases, and since Pasteurella wound infections are usually polymicrobial, recommended treatment is broad-spectrum such as amoxicillin-clavulanate. In isolated Pasteurella infections, first line treatment is penicillin, although there are some that favor testing isolates from sterile sites for the presence of beta-lactamase production, and treating those infections with ampicillin-sulbactam, pipercillin-tazobactam, or ceftriaxone. (Weber)

References

  1. Weber, David J., and Sheldon L. Kaplan. “Pasteurella infections.” UpToDate, Wolters Kluwer, 15 June 2018, http://www.uptodate.com/contents/pasteurella-infections?search=pasteurella%20treatment&source=search_result&selectedTitle=1~25&usage_type=default&display_rank=1#H14. Accessed 4 Feb. 2020.

-Tom Koster, DO is a 1st 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.

Respiratory Protection in the Days of the Novel Coronavirus

In the peak of the flu season we might see many people wearing masks in physician offices and hospitals. In the news today, as the 2019 Novel Coronavirus (SARS-CoV-2) continues to spread, we see many images of people wearing different types of respirators, some are N95 respirators and others are surgical masks. Not all experts agree on the efficacy of these pieces of personal protective equipment in the face of viruses, but if you’re going to use them, it is important to know how, when and why.

OSHA’s Respiratory Protection standard (1910.134) provides information about requirements for staff who may potentially be exposed to airborne pathogens. These requirements include specific instructions for choosing the proper respirator, for providing fit-testing, and for user training. The College of American Pathologists (CAP) also expects labs to determine the risk of airborne pathogen exposure for each employee, and they require labs to have a plan which outlines engineering and work practice controls that reduce exposure potential.

The purpose of a respirator is to protect the employee from contaminated or oxygen-deficient air. Therefore, two classes of respirators are common; air-purifying respirators which use filters to remove contaminants from the air you breathe, and atmosphere-supplying respirators which provide clean air from an uncontaminated source. These types of respirators can also be classified further as tight-fitting or loose-fitting.  Tight-fitting respirators need a tight seal between the respirator and the face and/or neck of the user in order to work properly. For now, let’s focus on the air-purifying respirators which are in high demand these days as a potential 2019-nCoV pandemic looms.

In the laboratory, N95 respirators are probably the most commonly-used respirators, often used for protection against tuberculosis and other airborne pathogens. These respirators filter out 95% of airborne pathogens that are 0.3 microns or larger. While the exact size of the 2019-nCoV is not yet known, most coronaviruses are slightly larger than 0.1 microns. Does that mean a N95 respirator (recommended by the CDC) will not offer protection from the coronavirus? Not necessarily.

According to biosafety expert Sean Kaufman (www.saferbehaviors.com), the filter in the N95 respirator works three ways- through interception, impaction, and diffusion. Interception collects larger particles which are blocked by mask fibers, and impaction collects larger particles which have too much inertia to be moved around the filter fibers. Diffusion occurs as smaller particles are bombarded with larger air molecules and are pushed against filter fibers. Most of the bacteria or virus particles are removed from the airstream making the respirator quite useful and protective (HEPA filters on a Biological Safety Cabinet work in much the same way).

Employees who may need to wear a tight-fitting respirator as part of their job are required to have fit-testing every year. This is required by OSHA, and contracted employees (such as pathologists) should be fit-tested as well. Employees who may need such respirators would be those who work in microbiology labs, cytology techs who participate in patient procedures, and others. Labs should perform a risk assessment for each job category to determine the type and level of potential harmful airborne exposure.

Procedure masks, such as those handed out when people suspect they have the flu, are not technically considered respirators. Often, the person who is sick will wear these masks in order to prevent the spread of droplets when coughing or sneezing. They can protect others in the area, but they do not protect the user from harmful airborne pathogens or vapors.

Can these surgical masks be useful for the healthy public when a coronavirus is present? Sean Kaufman says “yes. If you wear a surgical mask in a potentially contaminated environment (on a commuter bus, for example),” Kaufman says, “it can keep you from touching your nose or mouth- two major routes of entry for viruses. Behaviorally speaking, these masks do offer some protection.”

Knowing when and why you use a respirator is vital, but knowing how to use it is important as well. Tight-fitting respirators should never be used without fit-testing to make sure the correct size is being used. Otherwise, the protection offered will be limited. Make sure your staff is properly trained and protected to work in environments where the air is not safe to breathe, and teach others about the usefulness of respirators when the flu and other viruses are lurking!

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.