Lablogatory

The Latest Pox, Up Close and Personal

Against the backdrop of COVID-19, the world experienced a multicounty outbreak of Mpox (formally monkeypox) beginning in May of 2022. Prior to that time, the virus was primarily known to circulate within central and west African nations causing zoonotic disease. Clinical presentations of Mpox comprise signs and symptoms including rash on the hands, feet, face or mucous membranes and patients may experience fever or an influenza-like illness.¹ Historically, transmission was associated with travel to an endemic region and contact with an infected animal. Importantly, the outbreak in 2022 was associated with broad changes in Mpox epidemiology, as most infections were acquired via sexual transmission.

Pox viruses and Mpox

Pox viruses are members of the family Poxviridae, which are double stranded DNA viruses that replicate entirely in the cytoplasm of host cells. They have worldwide distribution and cause disease in humans and other animals. Infection typically manifests as the formation of lesions, skin nodules or rash. Mpox belongs to the genus Orthopoxvirus which also includes other clinically important viruses including variola virus (smallpox), vaccinia virus, and cowpox. In the context of diagnosis, differentiation between the members of the Orthopox family becomes important.

The duration of illness with Mpox is usually between 2-4 weeks, with a variable incubation time most often between 6-13 days. The Mpox rash has historically been more focused on the face and extremities,² and will cycle through stages including encrustation, scabbing, and eventually resolution. During the 2022 outbreak, an increasing number of presentations involved the anogenital and oral regions, further highlighting the change in epidemiology. The window for transmission is currently an area of active research as new data suggests transmission can begin prior to the appearance of symptomology.³

Diagnosis – Molecular

Mpox is generally diagnosed using PCR testing from a swabbed lesion. At the onset of this emerging infectious disease, the CDC shared its algorithm and testing for Mpox with public health laboratories. The first-generation algorithm largely reflected its potential use as a tool for screening for bioterrorism agents, which included using two-tiered testing. The first test was designed to demonstrate that Orthopox DNA was present and rule out variola virus by targeting the Orthopox DNA polymerase gene found not present in Variola (E9L-NVAR). The second step was to target an Mpox-specific gene encoding the envelope protein (B6R).⁴ It soon was readily apparent that the only Orthopox virus in circulation was Mpox, so the CDC updated its guidance in late June 2022 to confirming diagnosis of Mpox with the single Orthopox DNA-polymerase PCR assay.

However, despite this modification to improve expediency and like the situation faced at the onset of the COVID-19 pandemic, the need for testing greatly exceeded what public health infrastructure could support. Thus, laboratories designed and validated laboratory developed tests (LDTs) to expand access to testing, thus enabling physicians to interrogate the causes of a patient’s rash more thoroughly. This flexibility was essential given rising cases numbers and relatively non-specific symptomology of Mpox. By May 2023, over 80 laboratories registered Mpox LDTs with the Food and Drug Administration,⁵ and commercial device manufacturers are now including it in new and forthcoming assays still in development.

Diagnosis – Histopathology

Although PCR testing is the mainstay of diagnosis, histopathologic evaluation of biopsy material from a lesion can also provide insight into the viral etiology. Mpox infected skin biopsies demonstrate similar histopathologic features of infections caused by other pox viruses. As the rash continues to evolve over time, representative histopathological changes can also be observed. Early lesions may demonstrate ballooning degeneration, acanthosis and spongiosis. More mature lesions progress to near total keratinocyte necrosis with exocytosis comprised of mixed cellular inflammatory infiltrate.⁶ Eosinophilic bodies may be identifiable in the cytoplasm of infected cells, commonly known as Guarnieri bodies, represent the mature virions produced in the cytoplasm of infected cells.

Recently, the histopathological description of 20 outbreak-associated clinical cases of Mpox from Spain was reported. Epidermal necrosis and keratinocytic ballooning were commonly encountered microscopic features associated with Mpox lesions.⁷ Figure 1 is a skin biopsy from a patient who presented with a vesicular eruption in September with a history of mpox, syphilis and herpes simplex infection whose lesions were worsening. It similarly shows ballooning degeneration, epidermal necrosis, exocytosis of neutrophils into the epidermis, and intracytoplasmic eosinophilic inclusions (Guarnieri bodies) (Figures 2-3).

Figure 1. Histopathology of MPOX from a biopsied skin lesion (4x magnification, H&E). Intact epidermis with evidence of ballooning keratinocyte degeneration and infiltration of neutrophils.

Figure 2. Histopathology of Mpox (10x magnification, H&E). Epidermis with a cross-section of follicular infundibulum (hair follicle) is in the bottom left. The keratinocytes to the right demonstrate marked vacuolar change and small eosinophilic bodies can be observed in a background of neutrophils and necrotic keratinocytes.

Figure 3. Histopathologic findings of MPOX in a biopsy of a skin lesion (40x magnification, H&E).
High power magnification of viral inclusions, guarnieri bodies, (arrowheads) in a background of necrotic keratinocytes and neutrophilic infiltrate.

Treatment

Mpox is much milder than smallpox despite similar rash manifestations. In cases of severe Mpox infection, therapies used for smallpox have been compassionately utilized, but supportive measures are the mainstay of management of uncomplicated cases. Vaccination is now available as both a pre-exposure prophylaxis and post-exposure prophylaxis. It is important to note that the clinical effectiveness of the currently used vaccine in the United States is not known; however, early data across 32 US jurisdictions showed that among males 18-49, those who were unvaccinated had an Mpox incidence 14 times higher than similarly aged males who received at least one dose of vaccine at least 2 weeks prior.⁸

Conclusion

The Mpox outbreak, declared a global health emergency in July of 2022, has reinforced the need for flexibility within laboratories and industry to respond to emerging infectious diseases. The global health emergency for Mpox was declared over on May 11, 2023, but cases are still going to sporadically occur and minor outbreaks will result. The rapid development of numerous LDTs was essential to support the overwhelmed public health infrastructure, and this continued flexibility is needed to appropriately respond to future public health emergencies.

References

https://www.cdc.gov/poxvirus/mpox/symptoms/index.html. Accessed April 19th, 2023.
Saxena et al. J. Med. Virol. 2022;95:e27902. DOI: 10.1002/jmv.27902
https://www.cdc.gov/poxvirus/mpox/about/science-behind-transmission.html Accessed May 19th, 2023
Li Y, Olson VA, Laue T, Laker MT, Damon IK. Detection of monkeypox virus with real-time PCR assays. J Clin Virol. 2006 Jul;36(3):194-203. doi: 10.1016/j.jcv.2006.03.012. Epub 2006 May 30. PMID: 16731033; PMCID: PMC9628957.
https://www.fda.gov/medical-devices/emergency-situations-medical-devices/monkeypox-mpox-and-medical-devices#Laboratories. Accessed May 3, 2023.
Bayer-Garner IB. Monkeypox virus: histologic, immunohistochemical and electron-microscopic findings. J Cutan Pathol. 2005 Jan;32(1):28-34. doi: 10.1111/j.0303-6987.2005.00254.x. PMID: 15660652.
Rodríguez-Cuadrado FJ, Nájera L, Suárez D, Silvestre G, García-Fresnadillo D, Roustan G, Sánchez-Vázquez L, Jo M, Santonja C, Garrido-Ruiz MC, Vicente-Montaña AM, Rodríguez-Peralto JL, Requena L. Clinical, histopathologic, immunohistochemical, and electron microscopic findings in cutaneous monkeypox: A multicenter retrospective case series in Spain. J Am Acad Dermatol. 2023 Apr;88(4):856-863. doi: 10.1016/j.jaad.2022.12.027. Epub 2022 Dec 26. PMID: 36581043; PMCID: PMC9794029.
https://www.cdc.gov/poxvirus/mpox/clinicians/vaccines/vaccine-considerations.html. Accessed May 3, 2023.

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

-Travis Vandergriff, MD is an Associate Professor and Board-Certified Dermatopathologist and practicing Dermatologist at UT Southwestern Medical Center.

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

Toxicology and Forensic Pathology: More Than a Numbers Game

I was recently reviewing new toxicology reports from my pending autopsies, and came across a report with the following results:

Looking at this in isolation, it would be easy to assume this person died from an overdose. Even low levels of fentanyl can be dangerous to an opioid-naive individual – a level this high is rare. Then there’s the added presence of fluoro fentanyl, a fentanyl analog, which would seem to support the notion of an overdose. The problem with this assumption? This person died from blunt force trauma as a pedestrian struck by a car. He was, according to witness accounts, walking and talking right until the moment of impact. Autopsy had shown multiple blunt force injuries incompatible with life.

This situation illustrates some of the complexity of postmortem forensic toxicology. Despite methodology being nearly the same, toxicology in a forensic setting differs in many important ways from that performed in a clinical setting.

The first major difference occurs in the pre-analytical phase. The results of clinical testing may be used to alter therapy or make a diagnosis. However, forensic toxicology results are meant to be used in a court of law, meaning the chain of custody needs to be maintained. If there is no documentation of who touched the sample and when, the integrity of the specimen can be called into question and results may be impermissible.

Not all forensic toxicology is performed on deceased patients. Specimens may be taken from the living during evaluation of an alleged assault, driving under the influence, or for workplace monitoring. In autopsy specimens though, postmortem redistribution (PMR) is another pre-analytical factor to consider. After death the stomach, intestines, and liver can serve as a drug reservoir and passively transfer the drug to surrounding vasculature. Other organs can also act as reservoirs, depending on where the drug is concentrated in life. Drugs which are highly lipid-soluble and/or have a high volume of distribution will diffuse down their gradient from adipose tissue into the bloodstream – antidepressants are notorious for this, and elevated postmortem levels need to be interpreted with caution.

Autopsy specimens are also more varied in type and quality than typical clinical specimens. Vitreous fluid, bile, and liver tissue are commonly collected at autopsy, in addition to central (heart) and peripheral (femoral or subclavian) blood. Femoral blood vessels, being relatively isolated from PMR-causing drug reservoirs, are a preferred source of specimens. Decomposition or trauma can limit the types or quantity of specimens and may even alter results. After death, bacteria from the GI tract proliferate and can produce measurable levels of ethanol in the blood. Decomposition also produces beta-phenethylamine, which can trigger a ‘positive’ result for methamphetamine on ELISA-based tests.

The post-analytical phase of autopsy toxicology also poses unique challenges. Lawyers and law enforcement will sometimes ask what the ‘lethal level’ of a drug is, and they’re invariably disappointed by my response. While there are published ranges of toxicity and lethality for most drugs, these are only general guidelines. There is no absolute lethal blood level for prescription or illicit drugs. Opioid users develop tolerance, making them relatively immune to a dose which would kill an opioid-naive person. In the example of the pedestrian described above, he had a long history of heroin abuse and could therefore tolerate much higher levels than most. For stimulants like cocaine and methamphetamine, there are no documented ‘safe’ levels as any amount could act as an arrhythmic agent. To add to the complexity, most overdose deaths involve multiple substances which may have synergistic effects and interactions that are difficult to parse.

Because of the reasons given above, the National Association of Medical Examiners still recommends full autopsy for possible overdoses. Deciding if a death was due to overdose is more complex than just reading a toxicology report – it requires interpretation and correlation with the autopsy findings and overall investigation.

References

D’Anna T, et al. The chain of custody in the era of modern forensics: from the classic procedures for gathering evidence for the new challenges related to digital data. Healthcare. 2023 Mar;11(5):634.

Davis GG, et al. National Association of Medical Examiners Position Paper: Recommendations for the Investigation, Diagnosis, and Certification of Deaths Related to Opioid Drugs. Acad Forensic Pathol 2013 3(1):77-81.

Pelissier-Alicot AL, et al. Mechanisms underlying postmortem redistribution of drugs: A review. J Anal Toxicol. 2003 Nov-Dec;27(8):533-44.

You Can’t Hide Those Safety Eyes!

Jamie, the manager of a large metropolitan hospital lab, has many responsibilities. She must spend most of her time in the office, on the phone, or in meetings. She does find time to come out to speak with the employees, but only for a second to check on things or maybe make a request. During a recent safety audit, Jamie received feedback that several employees were seen working in the lab without using the proper PPE. One tech was working the bench without gloves, one individual had their lab coat on but not buttoned, and one auditor noticed that no one in the lab was wearing face or eye protection. This came as a shock to Jamie, she had never noticed this before. This doesn’t necessarily mean that Jamie is a bad manager, it could be that she was so focused on daily operation issues and she failed to notice other problems.

We have all heard the term “nose blind.” It’s when a person is around a bad smell so frequently that they become oblivious to its presence, and this can actually happen with vision as well. Have you ever heard the phrase, “you can’t see the forest for the trees,” or maybe the term “snow blind?” This phenomenon occurs when someone is concentrating so hard on one problem they may miss a more serious safety issue directly in front of them. Lucky for us, we have a tool to help those safety issues stand out. We have our “Safety Eyes!”

Ok, so what exactly are Safety Eyes? Are they some kind of new eye protection device that fit directly on your eyes? Are they indestructible eyes? Not exactly. Safety Eyes is a term used to describe the ability to spot current or potential safety issues more easily. It is the ability to walk into a room and immediately scan the environment for safety issues. This ability doesn’t just magically develop, it takes time and effort to master, and once you have it, you will begin to notice issues without even trying.

There are methods you can use to develop your safety eyes. Like any other sense, it is important to practice using it frequently so that its use becomes second nature to you. Think about this in terms of a wine sommelier. A sommelier may train for several years to acclimate their nose and palate in order to detect various nuances in different types of wine. It is through experience and exposure to many different types of wine that they are able to pick up on the slightest hint of a flavor or scent. This same repeated exposure works for sharpening your Safety Eyes as well. It is probably unlikely that you have a Safety Unicorn in your lab who can pick up on potential safety issues on their first day on the job. To become better at seeing safety issues, perform periodic rounding in the department and look for specific safety issues. Start by covering one specific safety area such as PPE use, waste management or fire safety. Your ability to quickly notice issues in these areas will sharpen, and you will be able to expand your newly honed power to other areas.

By developing your Safety Eyes, you will become more aware of various types of safety issues and where they are most likely to be encountered. It is easy to become “nose blind” to safety issues in a lab where you work every day. Start by simply using a checklist to focus specifically on one new safety area and soon the issues that may have been there all along will be more easily detected. Now that you can see the forest, you can make those important changes which will improve your overall lab safety culture!

-Jason P. Nagy, PhD, MLS(ASCP)^CMis a Lab Safety Coordinator for Sentara Healthcare, a hospital system with laboratories throughout Virginia and North Carolina. He is an experienced Technical Specialist with a background in biotechnology, molecular biology, clinical labs, and most recently, a focus in laboratory safety.

Thoracic Aortic Disease at Autopsy: An Opportunity for Intervention

When I perform an autopsy, I know that anything I find will be discovered too late to save my patient’s life. I generally hope the autopsy report helps explain why and how someone died to family members, law enforcement, lawyers, and potential jurors. It’s less common that an autopsy finding can have immediate impact on the healthcare of the decedent’s family, but this is the case with thoracic aortic disease.

Thoracic aortic aneurysms and/or dissections (TAAD) are relatively common mechanisms of sudden, unexpected, and natural death we encounter in forensic pathology. The classic teaching is that TAAD are caused by hypertension, bicuspid aortic valves, pregnancy, and cocaine use. Genetic disease^1,2,3is included late in the list, almost as an afterthought, with Marfan syndrome given as the prototype. However, the evidence for a genetic underpinning of TAAD has been steadily expanding. Family pedigrees in the late 1990s revealed up to 20% of patients with TAAD had an affected first-degree relative.^4,5 If extended to 2^nd degree relatives, up to 43% of patients with TAAD had at least one affected family member.⁶ There are also now more than 15 types of defined connective tissue diseases, and more than 29 genes identified which are mutated in heritable TAAD (H-TAAD). Clearly, the etiologies of hereditary TAAD (H-TAAD) expand far beyond Marfan syndrome.

H-TAAD can be categorized as “syndromic” or “non-syndromic” and has wide variability in clinical presentation. Syndromic forms show multisystem involvement, and often have externally apparent phenotypes. The most common forms of syndromic H-TAAD are Marfan syndrome, vascular Ehlers-Danlos, and Loeys-Dietz syndrome. However, the physical appearances associated with these syndromes can be subtle and aren’t always present.⁷ Non-syndromic H-TAAD affects only the aorta and aortic valve and includes both bicuspid aortic valve-associated TAAD and “familial” H-TAAD.

Thoracic aortic disease often remains subclinical until serious, life-threatening complications occur. Forensic pathologists can therefore play an important role in preventative health, proactively identifying patients at high risk for TAAD. Because a relatively large percentage of thoracic aortic disease is hereditary, it is prudent to ask whether forensic pathologists should initiate genetic testing in these situations. Unfortunately, postmortem genetic testing is still out of reach for most Medical Examiner and Coroner systems. Insurance companies don’t reimburse for postmortem genetic tests, even when requested by healthcare providers of the surviving family. Selective testing of decedents with high-risk features may be more affordable, but there is no consensus on what those “high-risk” features are – and because of the variable penetrance and expressivity of H-TAAD, patients can present at nearly any age (even in their 80s).^5,6

Despite the limitations of access to postmortem genetic testing, the most practical resource forensic pathologists have at our disposal is the telephone. According to 2022 guidelines from the American College of Cardiology and American Heart Association, aortic imaging is recommended in all first-degree relatives of patients with TAAD to screen for occult disease.⁸ Family members therefore need to be notified of this recommendation, regardless of the pathologist’s choice to pursue genetic testing. Developing a collaborative relationship with a local hospital or academic center may be beneficial; medical examiner offices then have a place to refer families for screening, and genetic counselors in these locations are better suited to evaluate the entire family and potentially order targeted genetic tests.⁹

Forensic pathologists are in a crucial position to recognize potential H-TAAD. Surgical outcomes for patients with TAAD are much better when performed prophylactically rather than emergently, so identifying aortic disease prior to rupture or dissection is crucial. Premature attribution to hypertension, or overreliance on the presence of Marfan-like features to identify hereditary disease, will result in missed opportunities to save lives. Forensic pathologists can therefore have a significant impact on public health, by recognizing the heritability of TAAD and contributing to improved screening of families at risk.

References

Prahlow JA, Barnard JJ, Milewicz DM. Familial thoracic aortic aneurysms and dissections. J Forensic Sci. 1998 Nov;43(6):1244-1249.
Gleason T. Heritable disorders predisposing to aortic dissection. Semin Thorac Cardiovasc Surg. 2005 17:274-281.
Gago-Diaz M, Ramos-Luis E, Zoppis S. Postmortem genetic testing should be recommended in sudden cardiac death cases due to thoracic aortic dissection. Int J Legal Med. 2017 Sep;131(5):1211-1219.
Biddinger A, Rocklin M, Coselli J, et al. Familial thoracic aortic dilatations and dissections: a case control study. J Vasc Surg. 1997;69:506-511.
Coady MA, Davies RR, Roberts M, et al. Familial patterns of thoracic aortic aneurysms. Arch Surg. 1999;134:361-367.
Chou AS, Ma WG, Mok SCM, et al. Do familial aortic dissections tend to occur at the same age? Ann Thorac Surg. 2017 Feb;103(2):546-550.
Isselbacher EM, Cardenas CLL, Lindsay ME. Hereditary influence in thoracic aortic aneurysm and dissection. 2016. Circulation 133(24):2516-2528.
Isselbacher EM, Preventza O, Hamilton Black 3rd J, et al. 2022 ACC/AHA Guideline for the diagnosis and management of aortic disease: A report of the American Heart Association/American College of Cardiology Joint Committee on Clinical Practice Guidelines. Circulation. 2022 Nov 2. Online ahead of print.
Krywanczyk A, Rodriguez ER, Tan CD, Gilson T. Thoracic aortic aneurysm and dissection: Review and recommendations for evaluation. Am J Forensic Med Pathol. 2023 Mar 6.

-Alison Krywanczyk, MD, FASCP, is currently a Deputy Medical Examiner at the Cuyahoga County Medical Examiner’s Office.

Microbiology Case Study: New Presentation of Chest Pain and Cough

Case History

A 35 year old male presented to the emergency department with left-sided chest pain, a new cough and pain that gets worse with inspiration. The past medical history was significant for HIV, recurrent syphilis, rectal high-grade dysplasia, proctitis with possible rectal abscess with concomitant Herpes Simplex Viral infection and Cytomegalovirus colitis. This patient was not compliant with antiretroviral therapy (ART) and his CD4 count was <100 at admission. In the ED, a chest X-ray was performed which revealed bilateral patchy, peripheral mass-like opacities with ground glass halos (Figure 1). Routine respiratory cultures of sputum and bronchoalveolar lavage (BAL) revealed no significant observations; mainly respiratory flora was reported. However, hematology staining of BAL revealed multiple fungal elements consistent with Pneumocystis jirovecii organisms (Figure 2).

Bilateral patchy airspace opacities with a loculated left pleural effusion (Figure 1, top). Wright Giemsa staining of *Pneumocystis jirovecii* cysts (with intracystic bodies, dots in the cysts) in a bronchoalveolar lavage specimen (Figure 2, bottom).

Discussion

Previously classified as a protozoa, Pneumocystis jirovecii (formerly known as Pneumocystis carinii) is currently considered a fungus based on nucleic acid and biochemical analysis. Stringer et al. proposed the change in nomenclature in early 2002, in honor of the Otto Jirovec, who was credited by some, as the original descriptor of the organism in human hosts.¹ A few Pneumocystis species have been described in a wide variety of mammalian hosts but P. jirovecii is only capable of infecting humans and is not capable of infecting other animals.⁴P. jirovecii infection mostly affects the immunocompromised patients and can lead to severe, life-threatening disease. HIV is one of the most commonly encountered underlying diseases, but individuals with cancer, transplant recipients and hosts receiving immunosuppressive medication can be at risk. The route of transmission is thought to be person to person through air transmission. Immunocompetent hosts can act as reservoirs of the organisms and spread it to the immunocompromised. The use of antiretroviral therapy and prophylactic medication on HIV patients has substantially decreased the incidence of PJ infection in this population.^{2, 3}Defects in cellular immunity, specifically CD4+ T-cell-mediated immunity is a predisposing factor for the development of severe Pneumocystis infection. The disease is usually a pneumonia that can have a slow progression or progress rapidly. Fever, nonproductive cough, tachypnea, and severe dyspnea with hypoxia are the most common symptoms.

Induced sputum, bronchoalveolar lavage fluid, or lung tissue are the commonly accepted specimens received in clinical laboratories for direct diagnosis of P. jirovecii. Microscopically, the life cycle of Pneumocystis consists of at least two different life cycle forms of Pneumocystis organisms: the trophic form and the cyst form. The trophic form generally measures ∼2 µm in greatest diameter and in contrast, the cyst is significantly larger, measuring ∼8–10 µm in greatest diameter. The rigid Pneumocystis cyst wall is formed of β-glucan, which warrants detection of systemic Pneumocystis infections using Fungitell testing.⁴Trophic and cyst forms can be detected with Papanicolaou, Gram-Weigert, or Wright Giemsa, Gomori methenamine silver (GMS), or calcofluor white. The sensitivity of the interpretation of these stains depends upon the expertise of the observed to differentiate Pneumocystis from artifacts and other fungi such asand Histoplasma capsulatum. The use of monoclonal antibodies with Immunofluorescent antibody stains directed against human Pneumocystis epitopes, can enhance direct detection of this organism in clinical specimens.⁵

Because the sensitivity of special stains and the microscopy depends, in part at least, on the experience and skill of the microscopist, polymerase chain reaction (PCR) has become a newer and promising testing method for P.jirovecii DNA detection. It is recommended that PCR should be done in cases with only mild to moderate immunosuppression because these individuals may have a lower burden of the fungus and clinical and radiologic findings are less developed compared to severely immunosuppressed patients.⁶Another advantage of PCR is the ability to quantify the amount of P. jirovecii in the specimen, which has been suggested to help distinguish individuals who may be colonized versus those with true P. jirovecii associated pneumonia.⁷

The drug of choice for prophylaxis against or treatment of P. jirovecii is trimethoprim-sulfamethoxazole. Prophylaxis may generally be started on HIV positive patients once the CD4+ is <200cells/microL, CD4% is <14%, and/or patients have a detectable viral load.⁸

–Ilianne Vega Prado is a PGY-3 pathology resident at George Washington University. She has worked as a Microbiology technologist for a few years before completing her medical degree. Her academic and research interest include antibiotic stewardship, cytopathology, and breast pathology.

-Rebecca Yee, PhD, D(ABMM), M(ASCP)^CM is the Chief of Microbiology, Director of Clinical Microbiology and Molecular Microbiology Laboratory at the George Washington University Hospital. Her interests include bacteriology, antimicrobial resistance, and development of infectious disease diagnostics.

Thyroid Tales (First Edition)

I’ve found that our cytologists have a love-hate relationship with thyroids. Pathologists do too. Or it could be that we see so many goiters (50%) and follicular lesions or atypia of undetermined significance (35%) that the rare papillary thyroid carcinoma is a gem in our eyes. Minimally-invasive thyroid FNAs are instrumental in the management of thyroid nodules. It’s important to note that due to lack of architecture and assessment of capsular invasion, cytologic diagnoses may be limited, and prior to referring the patient for a potentially unnecessary surgery, various molecular tests can be utilized. The ongoing evolution of molecular testing on thyroid FNAs help classify indeterminate and suspicious cytology diagnoses (Bethesda Categories III and IV), examining the risk of malignancy or detecting the presence of genetic alterations, which help guide surgical intervention versus surveillance. This post (the first edition) features a series of our classic Bethesda Category VI specimens, which bypassed the need for risk classification and defaulted in surgical intervention based on guidelines at the time of diagnosis. It is worth mentioning that many of these cases occurred prior to the implementation of the Thyroid Imaging Reporting and Data System (TI-RADS), so to preserve the accuracy of patient history, a TI-RADS score will not be assumed.

Case 1

Okay, I know I said Bethesda VI, but let’s kick this series off with a Bethesda Category IV case. Thankfully, the patient decided to undergo a partial thyroidectomy, yielding a beautiful tissue follow-up. A 59-year-old male with newly diagnosed melanoma of the neck underwent imaging for staging purposes. A left thyroid nodule was identified measuring 3.0 centimeters. The patient presented for an ultrasound-guided fine needle aspiration.

Images 1-3: Thyroid, Left Lobe, FNA 1: DQ-stained smear; 2: Pap-stained smear; 3. H&E Cell Block section (400X).

Abundant oncocytic Hürthle cells, some with mild atypia, were identified, suggestive of Hürthle cell neoplasm (Images 1-3). With a lack of lymphocytes, we did not feel comfortable suggesting Hashimoto’s (lymphocytic) thyroiditis. Immunostains performed on cell block sections show the tumor cells are positive for TTF-1, focally positive for thyroglobulin and AE1/AE3 (rare), and negative for calcitonin. The morphology and immunohistochemical profile support the above diagnosis.

The patient underwent a left lobectomy and isthmusectomy. Pathology showed a 3.2 cm Hürthle cell carcinoma (Images 4-5) in the left lobe of the thyroid (encapsulated with a foci of capsular invasion without vascular invasion) as well as a 0.3 cm micropapillary carcinoma. Since Hürthle cell carcinoma does not typically concentrate radioiodine, the patient would not be responsive to treatment with radioactive iodine. Therefore, there would be less benefit derived from treating the smaller right lobed nodules (which don’t meet biopsy criteria) from remnant ablation. The patient had a clinically limited stage thyroid cancer. The patient is monitored with neck ultrasounds rather than serum thyroglobulin testing (due to the remaining right lobe).

Images 4-5: Thyroid, Left Lobe with Isthmus, Excision: H&E section (600X).

Cytology diagnosis: Hürthle cell neoplasm.

Pathology diagnosis: Hürthle cell carcinoma.

Case 2

A 53-year-old female with no prior history presented with fatigue and a self-palpated right thyroid nodule and normal thyroid function tests. She reported an extensive family history of hypothyroidism. On thyroid ultrasound, the right upper pole thyroid nodule measured 2.0 x 2.0 x 1.8 cm and was mostly solid and hypoechoic with microcalcifications. Pre-intervention serum calcitonin measured 1634 pg/mL. The patient underwent an FNA of the thyroid nodule and the smears are depicted below.

Images 6-7: Thyroid, Right Lobe, Upper Pole, FNA: 6: DQ-stained smear; 7: Pap-stained smear.

Cells appear plasmacytoid and appear both isolated and in clusters (Image 6). The nuclei are eccentrically placed, and the chromatin has a salt and pepper appearance akin to a neuroendocrine tumor (Image 7-8). Also identified were pink granules and intranuclear pseudoinclusions (Images 6-7). We performed immunohistochemical stains on paraffin sections of the cell block. Tumor cells show positive staining for calcitonin, chromogranin, mCEA, and TTF-1, while negative staining for thyroglobulin and CD45.

Following the diagnosis, the patient had a CT scan for staging purposes. Multiple lymph nodes in the right cervical chain were identified. the patient at a clinical stage IVA diagnosis. In the interim, the patient had a total thyroidectomy which revealed medullary thyroid carcinoma of the right lobe measuring 2.2 cm, a micropapillary carcinoma of the left lobe measuring 0.1 cm (Image 8). Lymphovascular invasion was not identified, the inked surgical resection margins are free of carcinoma, and metastatic medullary carcinoma was identified in 6 of the 77 lymph nodes removed during the central compartment lymph node dissection, and bilateral cervical lymphadenectomies. The calcitonin level dropped to 23 pg/mL postoperatively. Genetic testing was performed to assess for Multiple Endocrine Neoplasia Type 2 (MEN2), and although her result was indeterminate, a RET mutation was not identified.

Image 8: Thyroid, Excision: H&E section (600X).

Case 3

A 67-year-old male with no pertinent medical history presented to the endocrinology clinic after his primary care physician identified a large lump in the patient’s neck. A 7.0 cm hypoechoic right thyroid mass with macrocalcifications was noted on ultrasound imaging. The patient was referred to diagnostic imaging for a thyroid FNA. The smears and cell block section are depicted below. While the papillary formation of Image 9 is not evident on the pap-stained slide (Image 10), the nuclear grooves and pseudoinclusions along with irregular nuclear membranes and powdery chromatin are highlighted. A separate needle pass was collected for molecular testing, which revealed a BRAF V600E mutation in the tumor cells.

Images 9-11: Thyroid, Right Lobe, FNA 9: DQ-stained smear; 10: Pap-stained smear; 11. H&E Cell Block section (600X).

Two weeks after the FNA diagnosis, the patient was scheduled for a partial thyroidectomy of the right lobe. While 60% of the mass demonstrated well-differentiated papillary thyroid carcinoma (Image 12), 40% of the tumor contained poorly differentiated thyroid carcinoma with squamous features (Image 13). No sarcomatous components or giant tumor cells were identified. Carcinoma with squamous features invaded into the surrounding tissue, strap muscle, thymus, and right paratracheal lymph node. Interestingly, the right-sided levels 3 and 4 lymph nodes contained predominantly well-differentiated papillary thyroid carcinoma with rare foci of poorly differentiated thyroid carcinoma with squamous differentiation.

Images 12-13: Thyroid, Right Lobe, Excision: H&E section (600X).

Five months after the excision, the patient developed a left-sided pleural effusion. A diagnostic thoracentesis was performed and metastatic thyroid carcinoma was identified. Immunostains performed on the cell block slides with adequate controls show that the tumor cells are positive for PAX8, and negative for TTF-1, and thyroglobulin. The findings support the diagnosis. While patients with papillary thyroid carcinoma tend to have better disease-free survival rates, the poorly differentiated tumor was difficult to control and eventually resulted in widespread metastasis.

Cytology diagnosis: Papillary thyroid carcinoma.

Pathology diagnosis: Poorly differentiated thyroid carcinoma with squamous features in a background of well-differentiated papillary thyroid carcinoma.

Case 4

A 73-year-old female presented with a rapidly growing and painful thyroid mass that measured 8 cm on imaging. Originating from the right lobe, multiple needle passes targeted various areas of the mass via ultrasound-guidance. The smears and cell block section are presented below. Smears (Images 14-15) feature pleomorphic nuclei in a background of inflammation and necrosis. The cell block section (Image 16) demonstrates increased mitotic figures and neutrophils.

Images 14-16: Thyroid, Right Lobe, FNA: 14: DQ-stained smear; 15: Pap-stained smear; 16. H&E Cell Block section (600X).

We performed immunocytochemical stains on paraffin sections of the cell block. Tumor cells how positive staining for p53, focal staining for cyclin D1, and negative staining for AE1/AE3, thyroglobulin, and BCL-2. Rare tumor cells show staining for TTF-1. The proliferation index by Ki-67 immunostaining is approximately 70%.

While not a standard procedure for thyroid specimens, core biopsies (Image 17) were also obtained from this mass.

Molecular testing on the core biopsy sample identified a high mutation burden, with the tissue harboring both TP53-inactivating and TERT promoter mutations. Imaging demonstrated widespread metastasis, and this patient did not survive the extensiveness of her disease.

Cytology Diagnosis: Undifferentiated (anaplastic) thyroid carcinoma.

Pathology Diagnosis: High-grade carcinoma consistent with anaplastic carcinoma (interchangeable diagnoses).

That’s enough for our classic thyroid cases. Stay tuned for the second edition featuring thyroid FNAs with unsuspecting findings!

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

Microbiology Case Study: A 25 Year Old Male with Abdominal Pain and Fever

Case History

A 25 year old male with severe right lower quadrant abdominal pain, fever and history of congenital neutropenia and thrombocytopenia presented to the emergency department. The patient reported experiencing similar episodes over the previous six months, sometimes requiring hospitalization. His symptoms would typically improve following several days of antibiotic therapy and were thought to be secondary to typhlitis. He reported that his current symptomology was more severe than any of the previous episodes. In the emergency department, the patient was found to be tachycardic and severely hypotensive, prompting admission for sepsis and empiric antimicrobial therapy with piperacillin/tazobactam and vancomycin. Imaging at the time revealed severe terminal ileitis with mild pancolitis, markedly worsened from a study obtained months prior during a previous episode. After failing to improve with conservative management, an exploratory laparotomy with total abdominal colectomy and end ileostomy was completed to achieve source control.

Laboratory workup

Anaerobic bottles from blood cultures collected at the time of hospital admission flagged positive in under 24 hours. Gram staining from the positive bottle broth revealed large, gram variable bacilli (Image 1A). Multiplex PCR performed on the positive blood culture broth failed to reveal the identity of the organism. After 24 hours, close observation was required to visualize bacterial growth on anaerobic blood culture media, which appeared as a thin, translucent film (Image 1B). To provide better visualization for this case, a loop was utilized to scrape the growth so a path could be observed within the biomass (Images 1B and 1C, dotted lines). The bacterium recovered was definitively identified as Clostridium septicum by MALDI-TOF MS, prompting continued coverage with piperacillin/tazobactam until discharge.

Image 1. A) Representative Gram stain from the positive anaerobic blood culture bottle. Staining reveals large, gram variable rods. Given the size and morphology of the cells, a gram positive organism was suspected. B) Growth of the organism on CDC anaerobic blood agar after 72 hours. A lack of distinct colonies was noted, and the organism grew as a thin film across the surface of the plate which was difficult to visualize. The hatched oval denotes where a loop was pulled across the plate surface to help visualize the bacterial growth. A closer, enhanced image is presented in C.

Discussion

Clostridium septicum is an anaerobic, Gram-positive spore-forming bacillus.^1,2 Colony morphology on solid media evolves over time, with a characteristic “medusa head” morphology at under 8 hours, a “sand grain” appearance at approximately 24 hours, and visible swarming after 48 hours (Image 1B and 1C).² The organism is found in the soil and the gastrointestinal tracts of both humans and animals with the potential to cause a variety of diseases. It is especially problematic as an infectious agent of livestock and other ruminants, poultry, and horses.¹ Clinical manifestations of infection with C. septicum in humans includes necrotizing enterocolitis, bacteremia, and clostridial myonecrosis.² Prompt identification of infection with Clostridium septicum is essential due to the rapidly fatal nature of untreated bacteremia with this organism. The mainstay of treatment is early initiation of antibiotic therapy and aggressive surgical debridement of affected tissue. The drug of choice for medical management is penicillin, although metronidazole and carbapenems may be used in patients with penicillin allergies.

C. perfringens and C. septicum are the two clostridial species most associated with bacteremia in neutropenic patients.⁵ Clostridium septicum bacteremia can allow for the hematogenous dissemination of infection and can be rapidly fatal.³ Infection of the bowels of a susceptible host and compromise to the bowel wall integrity leads to entry of bacteria into the circulation. In severe cases, this in turn can lead to seeding of distal sites with progression to sequelae including clostridial myonecrosis (gas gangrene). While classical traumatic myonecrosis associated with an infected wound becomes contaminated with clostridial spores from the external environment (most commonly C. perfringens), atraumatic (or spontaneous) myonecrosis is associated with C. septicum (among other clostridial species) and results from hematogenous seeding of distal sites during bacteremia. Virulence and tissue necrosis are mediated by a variety of exotoxins produced by the organism,¹ with alpha-toxin being the primary virulence determinant.⁴

Importantly, Clostridium septicum bacteremia is strongly associated with both hematogenous and gastrointestinal cancers. For this reason, detection of this pathogen in the blood may warrant further patient evaluation for potential sources of underlying pathology.² In this patient’s case, typhlitis and neutropenic enterocolitis were precipitating factors leading to his C. septicum bacteremia as this patient presented without evidence of gastrointestinal malignancy. Following the procedure, the patient stabilized, and his condition continued to improve through recovery. His antibiotics were eventually discontinued, and he was discharged.

References

Alves MLF, Ferreira MRA, Donassolo RA, Rodrigues RR, Conceição FR. Clostridium septicum: A review in the light of alpha-toxin and development of vaccines. Vaccine. 2021;39(35):4949-4956. doi:10.1016/j.vaccine.2021.07.019
Mallozzi MJG, Clark AE. Trusting your gut: Diagnosis and management of clostridium septicum infections. Clinical Microbiology Newsletter. 2016;38(23):187-191. doi:10.1016/j.clinmicnews.2016.11.001
Koransky JR, Stargel MD, Dowell VR Jr. Clostridium septicum bacteremia. Its clinical significance. Am J Med. 1979;66(1):63-66. doi:10.1016/0002-9343(79)90483-2
Kennedy CL, Krejany EO, Young LF, et al. The alpha-toxin of Clostridium septicum is essential for virulence. Mol Microbiol. 2005;57(5):1357-1366. doi:10.1111/j.1365-2958.2005.04774.x
Bennet JE, Dolin R, Blaser MJ, Onderdonk AB, Garrett WS. Diseases Caused by Clostridium. In Mandell, Douglas, and Bennett’s principles and practice of infectious diseases (2^nd ed., pp. 2960-2968). Elsevier.

-Amanda Means is a PGY-1 resident in Anatomic and Clinical Pathology at the University of Texas Southwestern Medical Center in Dallas, Texas. Dr. Means did her undergraduate studies at Sam Houston St University in Huntsville, TX and received her medical degree from the University of Texas Health Sciences Center in San Antonio.

Containment and Contagion: How Far Do We Go?

One of my favorite older horror movies is The Shining. The acting, the music, and the pace of the story create a good long scare for the audience. In one scene, the mother and child are playing in an outdoor maze constructed of tall bushes. Later in the film they would be running for their lives in that selfsame maze, but I do not want to give away any spoilers if you haven’t watched it. The maze sometimes reminds me of some laboratory departments that I have seen, and I have seen how winding hallways and multiple doorways create confusion for lab employees and others in the facility.

The International Organization for Standardization (ISO) states in its standard Medical Laboratories — Requirements for Safety (ISO 15190:2020) that clean and dirty areas need complete separation from floor to ceiling. For example, the break room must be a completely separate room from the space where lab work is performed. While not all laboratories are ISO-certified, this is clearly an infection control and safety best practice. The Occupational Safety and Health Administration (OSHA) has been known to enforce this when they cite labs for incomplete separation of clean and dirty areas.

The Centers for Disease Control (CDC) published its resource, Biosafety in Microbiological and Biomedical Laboratories (BMBL-6th Edition) in 2020, and it requires a hand hygiene sink near the exit of laboratories so that hands can be washed before exiting the department. That means door handles inside the department must be considered clean and not touched with gloved hands.

Think about those requirements and apply them to your lab space(s). Do they work? Do they make sense to you? If you work in a Biosafety (BSL) 2 or 3 laboratory, you should understand the basics of pathogen containment. After all, the Biosafety Level of your laboratory is determined by the infectivity of pathogens present, the severity of disease they could cause, their transmissibility, and the nature of the work conducted. Based on risk and task assessments, labs should utilize the personal protective equipment (PPE) appropriate for the tasks performed, and that PPE should never be brought outside of the laboratory (another OSHA regulation).

One of the most common questions I receive from lab safety professionals is how to improve PPE compliance. The answer is, we need to make it easy for our staff to do the right thing. That means providing education so they understand the consequences of unsafe behaviors, and it means putting practices in place that make sense and are easy to follow.

In my travels I have seen labs that require that gloves must be removed before touching lab telephones and keyboards. I have seen specimen transport policies that allow for the use of wearing one glove for holding specimens and keeping the other hand clean to touch doors, and I have also seen staff who are told to keep a glove on to touch all lab door handles. If your staff are required to figure out that crazy maze of practices, they are not ever all going to get it right, and someone is going to acquire an infection.

If you require all gloves to be removed and hands to be cleaned before exiting the lab (as you should), are there sinks and waste cans at the exits? If lab coats must be hung up before going into the break room or rest room (as they should be), do you provide coat hooks near those doors? If staff must leave one BSL2 lab and walk through a clean hallway to deliver specimens to another lab, what do they wear? Do they wear their PPE (violating OSHA’s regulation) and do they touch door handles with gloves that other staff will use without gloves when leaving the department for the day?

Lab Acquired Infections (LAIs) are vastly under-reported, and many time people do not even realize how they got infected. There are probably many lab practices (like the ones mentioned above) that lead to these. Could this happen in your workplace? How can you prevent it?

Start, as always, with a risk assessment. Determine the hazards in your workplace, create hazard mitigation steps, and determine if any residual risks still exist. Take a good look at your physical layout. Can a “clean” hallway be designated as “dirty?” Would something as simple as adding a door to close off a hallway make it so safety regulations can be followed? Those risk assessments and diagrams used as a show-and-tell for facility leadership can often be what gets you the approval or funds you might need to make those changes. Correcting those unsafe practices and those long lab winding hallways and exits will certainly make it easy for staff to find the way in and out while containing the pathogens where they should be.

–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.

Microbiology Case Study: Infected Hangnail in a 13 Year Old Female

Case History

A 13 year old female presented to the emergency department with a painful left thumb swelling. Eight days ago, she picked out a hangnail from the medial side of her left thumb, resulting in a worsening pain and swelling. She went to urgent care five days ago and was prescribed Augmentin with no improvement. Physical examination showed a tender circumferential swelling of the right thumb past the flexion joint, with paronychia extending 1 cm in length with fluctuance extending an additional 0.5 cm. The wound was incised, and the drainage was sent to the microbiology laboratory for culture.

The culture grew Gram negative bacilli after 24 hours of incubation (Figure 1). The culture grew a moderate quantity of small nonhemolytic colonies that showed “pitting” appearance on blood (Figure 2) and chocolate (Figure 3) agar plates. The colonies notably smelled musty/bleachy. There was no growth on MacConkey agar.

Identification by Matrix-Assisted Laser Desorption Ionization – Time of Flight (MALDI-ToF) revealed Eikenella corrodens.

Discussion

Eikenella corrodens is a facultatively anaerobic, gram negative, oxidase positive, catalase negative, nonmotile, non-spore-forming, rod. It belongs to the HACEK group and is in the family Neisseriaceae. It is a part of the normal flora of the mouth and upper respiratory tract.

E. corrodens is an opportunistic pathogen that can lead to various infections including a variety of head and neck infections (ocular, mastoid, submandibular, thyroid), pleuropulmonary infections, skin and soft tissue infections, etc.). It can cause subcutaneous abscesses, cellulitis of soft tissues, and bacteremia in intravenous drugs users. E. corrodens also causes infections of the hand (i.e., ostomyelitis, cellulitis) because of chronic nail biting, human bite, or clenched-fist injury. The bacterium can gain access to the central nervous system by way of periodontal, middle ear, or sinus infections and cause meningitis, brain or spinal abscesses, subdural empyema, and osteomyelitis. In some predisposed individuals, endocarditis and bacteremia can also occur.

Under aerobic conditions, it grows slowly in blood and chocolate agar at 35–37℃ and does not grow on MacConkey Agar. It forms small, convex, round or irregular, gray, and non-hemolytic colonies. Most isolates may “pit” the agar although both pitting and non-pitting strains have similar biochemical characteristics. Most strains produce a musty or bleach-like odor.

E. corrodens is generally sensitive to ampicillin, amoxicillin, second- and third generation cephalosporins, tetracyclines, azithromycin, sulfamethoxazole, and fluoroquinolones. They are resistant to clindamycin, vancomycin, erythromycin, metronidazole, aminoglycosides, and penicillinase-resistant penicillins, with penicillin susceptibility varying from strain to strain. In clinical microbiology laboratories, the routine antimicrobial susceptibility testing for E. corrodens is not usually performed since it is generally responsive to beta-lactams. If necessary, susceptibility testing can be performed following the CLSI guidelines for fastidious organisms.

The disease spectrum of E. corrodens is increasing in complexity, and it is important to study its clinical characteristics for timely diagnosis, and effective treatment.

References

Chapter 9, Color Atlas and Textbook of Diagnostic Microbiology, Seventh Edition.
Li, L, Shi, Y-B, Weng, X-B. Eikenella corrodens infections in human: Reports of six cases and review of literatures. J Clin Lab Anal. 2022; 36:e24230. doi:10.1002/jcla.24230
Mühlhauser M. Eikenella corrodens. Rev Chilena Infectol. 2013. doi:10.4067/S0716-10182013000200007

-Fahad Sheikh is a 2^nd year AP/CP pathology resident in the Department of Pathology at Montefiore Medical Center in Bronx, NY.

-Phyu Thwe, Ph.D, D(ABMM), MLS(ASCP)^CMis Associate Director of Infectious Disease Testing Laboratory at Montefiore Medical Center, Bronx, NY. She completed her medical and public health microbiology fellowship in University of Texas Medical Branch (UTMB), Galveston, TX. Her interests includes appropriate test utilization, diagnostic stewardship, development of molecular infectious disease testing, and extrapulmonary tuberculosis.

The Importance of Patient Identification in Forensics

Body identification is one of the core responsibilities of a forensic pathologist yet is also probably the most common one to be overlooked. Most of the deceased people who come to our office are visually identifiable, and the identity may already have been confirmed if they were transported to the hospital. In some situations, though, the body may be disfigured by fire, decomposition, injury, or there may only be partial remains recovered. In incidents with multiple fatalities, we need to be sure the correct remains are returned to the correct family. Particularly when foul play is involved, there may be intentional attempts to conceal the decedent’s identity or disguise them as another person.

There are two different levels of identification: “positive” identification (the gold standard) and “presumptive” identification.

The three generally accepted forms of “positive” identification are DNA comparison, fingerprint comparison, and radiograph comparison. While television shows have DNA “matches” coming back in the time it takes for a commercial break, DNA identification can pose challenges. A pre-existing specimen from the decedent or close family members is needed for comparison, which means you need to already have some suspicion of who they are. If they’ve been previously arrested or charged with a felony (the laws vary slightly by state), their DNA may have been uploaded to the Combined DNA Index System (CODIS), and a match may potentially be obtained blindly by uploading the decedent’s sample. By comparison, one can collect fingerprints from a decedent and submit them to the Automated Fingerprint Identification System (AFIS) for relatively rapid identification. Many people have been fingerprinted in their lifetime, whether for relatively minor arrests, employment, or background checks. However, there are still limitations. The hands (or at least fingertips) need to be intact, with printable skin. For mummified remains, the tissue can be rehydrated by soaking in sodium carbonate or sodium hydroxide to obtain legible prints. “Degloved” remains, where the skin has sloughed from the hands due to decomposition, can be fingerprinted by inserting one’s gloved hand into the sloughed skin.

Radiographs, like DNA, are limited by the need to have a pre-existing sample from the decedent (meaning you need to know who they might be). Radiographs are invaluable when trying to identify someone with no usable fingerprints, or no fingerprints on file. A variety of locations can be used for comparison, including the dentition, frontal sinuses, vertebral processes, healed fractures, or orthopedic implants. Serial numbers on implanted devices can also be traced back to the decedent, although not all implantable devices have such markings.

“Presumptive” identifications are based on many other common sense factors including context, visual identification, tattoos, belongings, and clothing. Depending on the context of the case, a presumptive identification may suffice. For a decomposed body in a secure apartment occupied by a single, elderly person the neighbors haven’t seen in days, monogrammed dentures within the mouth may be sufficient. But in a fire with three charred female victims, aged 20-25, it’s much more important to confirm the identifications by a positive method. As I mentioned earlier any situation involving foul play may provide motivation for to conceal a victim’s identity, and so all homicide victims must be positively identified. It’s often taken for granted that the tag on our patient’s toe is accurate, but we need to approach our autopsies with the same level of diligence a laboratorian has when evaluating the label on a blood tube. Knowing who your patient is, and who your sample comes from, is the first critical step for any pathologist.