pathology – Page 2 – 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.

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.

The Basics of Deaths by Fire: Answering Your Burning Questions

Emergency services were called to a fire in a small apartment building, in which the structure was completely engulfed. Most of the occupants had been evacuated – however, once the fire was extinguished, the charred remains of an adult woman were found in the debris.

At the autopsy of severely fire-damaged human remains, two key questions must be answered: 1) who is the decedent?, and 2) were they alive when the fire started?

Question #1 is particularly relevant in this case, as many people lived in the building. Presumptive identification based on the tenant list may seem reasonable at first, but this victim could represent a visitor, contractor, or subletter. When facial identification isn’t possible, radiographic identification can be done with dental x-rays or x-rays of other bones which may have unique features from healed trauma or degeneration. Additional methods of positive identification could include fingerprints (if still intact), or DNA comparison to first degree relatives.

Question #2 is of importance because fire can be used in an attempt to disguise the identity of a victim of violent crime and destroy evidence. Cutaneous evidence of trauma may be disguised by burns, so full body x-rays are taken of every fire-damaged body. X-rays can also reveal retained bullets, knife tips, or fractures unlikely to have been caused by the fire.

When deciding if a fire victim was alive when the fire started, we first examine the upper and lower airways for soot. Most fire victims do not die from cutaneous burns, but from smoke inhalation – including carbon monoxide (CO) toxicity, which is often apparent by cherry red discoloration of the blood and viscera. Postmortem carboxyhemoglobin measurements in house fire victims are typically greater than 50%. There are exceptions to this rule, of course. Rarely, someone who was clearly alive when the fire began will have minimal or no soot in their airways and a negligible carbon monoxide concentration. This can happen in a “flash fire”, such as one ignited by gasoline or oxygen tanks, in which thermal injury to the upper airway may cause rapid occlusion by laryngospasm or edema. People with underlying heart or lung conditions will be more susceptible to the effects of carboxyhemoglobin, and may not survive long enough to obtain a level above 50%. Fires also produce other toxic products of combustion such as cyanide, and can lower ambient oxygen saturations to result in asphyxiation by lack of ambient oxygen (even without CO).

Forensic pathologists need to be aware of the artifacts that fires can create. Pugilistic posturing of fire victims (limb flexion) is due to heat-related contraction of muscle fibers. Epidural hematomas can result from boiling blood and bone marrow within the calvarium extravasating into the epidural space. The heat can induce fractures in exposed bone once the surrounding soft tissue is consumed or fully charred. Finally, the heat can split apart skin and soft tissue, resulting in sharp-force-like defects which occur parallel to the orientation of muscle fibers (rather than across them, which is more suspicious for penetrating trauma).

Of utmost importance in fire-related deaths, however, is scene investigation. The manner of death in fire fatalities is related to the origin of the fire. Most fire deaths are accidental, as the fire is unintentionally sparked by some electrical malfunction or unattended flame. However if the fire started intentionally, the manner of death can be homicide (if started by another) or suicide (started by the victim). It is therefore crucial to review the final fire investigation report before finalizing the autopsy report and death certificate.

This image shows dark black soot lining the main and lobar bronchi; this indicates the victim was breathing during the fire.

Heat-related epidural hematomas have a brown, amorphous appearance rather than the bright red color of traumatic epidural hematomas.

The scalp has been consumed by fire, and the exposed bone is calcined and brittle with fractures of the outer table.

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

Determining Time of Death: Separating Science from Pseudoscience

One of the most common questions I’m asked by family members is “do you know when they died?” If death occurs in the hospital, or is witnessed, the time of death isn’t controversial. It’s common though in forensics that people may not be found for hours, days, weeks, or more. Forensics television shows usually depict an investigator measuring body temperature at the scene, and then confidently declaring they’ve been dead for 44 hours. Unfortunately, there aren’t any existing methods that actually give that level of precision – but there is a way we can systematically approach the question.

When determining time of death (TOD), it’s most important to keep in mind that it will be an estimate. The estimate starts with the “window of death” – the time between when the decedent was last known alive and when their body was found. The smaller this window, the greater accuracy is possible.

Once the window is known, one can assess postmortem changes of the body. Livor mortis is the gravity-dependent settling of blood within vessels, which can appear as soon as twenty minutes after death. Sparing of lividity will be present in areas of pressure, such as parts of the body pressed against the floor or with tight clothing. Livor is initially blanchable, but after 8 to 12 hours blood extravasates from vessels and it becomes “fixed”. Clearly though, this only allows one to differentiate between ‘less than’ or ‘greater than’ 8 to 12 hours.

Rigor mortis (stiffening of the body after death) occurs because of postmortem ATP depletion. Muscle fibers require a supply of ATP to both contract and relax – once ATP levels are sufficiently low, muscle will remain contracted until the fibers are broken down by decompositional changes. Generally speaking, rigor starts to develop within an hour of death, peaks from 12 to 24 hours, and dissipates by 36 hours. However, these are average intervals. The onset of rigor is hastened by vigorous physical activity, seizures, electrocution, or increased body temperature, which preemptively deplete ATP. Rigor is also harder to detect in people with low muscle mass (e.g. infants), and can’t be assessed in frozen bodies with those with extensive thermal damage.

Cooling of the body after death, known as algor mortis, is similarly prone to interfering elements. One can find many formulas for estimating the time of death based on the temperature of the body – unfortunately, none of them are particularly useful because of the assumptions that must be made. Change in temperature after death is affected by numerous variables, including body habitus, clothing, wind, actual body temperature at the time of death (not many people are constantly at 98.6℉), sepsis, terminal seizures, and many others. If the environment is warmer than the body, the temperature can even increase after death.

I’ll briefly mention vitreous potassium measurement, which is probably the most recently discovered (and debunked) “holy grail” of time of death. Similar to algor and rigor mortis, vitreous potassium does a reasonably decent job predicting time of death in a controlled experiment – but in this line of work, people don’t tend to die in controlled environments.

At the end of the day, time of death is best estimated by thorough scene investigation, correlated with the evidence the body provides. Newspapers or mail not retrieved from the mailbox, expiration dates on perishable groceries, last refills of prescriptions, and unreturned text messages or phone calls can all narrow down the window of death.

As stated earlier, the longer the interval between death and discovery of the body, the more difficult time of death determination becomes. In advanced decomposition, there is no rigor, livor, or algor remaining to assess (there may even be scant residual soft tissue). In one such situation, despite months of a potential “window of death”, dates on unopened bills and crossed-off calendar dates helped us place the time of death within one or two days. It’s not as flashy as multivariate equations for temperature or potassium levels, but it’s far more accurate and scientifically defensible.

*Image 1. The quilting pattern of this decedent’s mattress is visible in the livor mortis on his back*.

Image 2. This decedent’s right arm is defying gravity due to rigor – he was initially face down, and his arm musculature became temporarily fixed in this position. Rigor can be forcibly broken if needed, but will also break down as decomposition proceeds.

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

Dying in a Winter Wonderland: Staying Safe as the Temperature Drops

A 40 year old man was found deceased in a parking garage in a Midwest city. It was late October and had rained the previous evening. He was identified by his sister who was a tenant in the adjacent apartment building. Unknown to her, he had recently been discharged from the hospital after a one-week psychiatric admission. His sister stated he was homeless and would occasionally sleep in the parking garage for shelter.

At the scene the decedent was prone on the ground, clad only in a pair of boxers. His water-soaked shoes, socks, sweatpants, and shirt were strewn about him. Autopsy revealed an atraumatic, thin adult man. Prominent pink discoloration was noted over the hips and knees. Internal examination showed only patchy black-brown discoloration of the gastric mucosa and pale kidneys. Histology was remarkable for subnuclear vacuolization of the renal tubular epithelium. The cause of death was certified as environmental hypothermia, and the manner of death accidental.

Hypothermia is defined as a core body temperature below 95℉ (35℃) and can result from endogenous illnesses like hypothyroidism or sepsis. The most common cause, though, is exposure to cold environments. On exposure, the hypothalamus initiates shivering and increases cellular metabolism to produce heat. Another crucial survival response is vasoconstriction, particularly of vessels in skin and skeletal muscle. If the overall loss of heat overtakes the body’s ability to produce or retain heat, hypothermia will result.

Developing hypothermia doesn’t require frigid weather – in dry air, temperatures of 50℉ can still result in hypothermia. Wind removes warmed air surrounding the body, and water conducts heat three times faster than air; therefore, with either of these factors present, people can develop hypothermia at even warmer temperatures,

The autopsy findings of hypothermia are not specific. External examination may show bright pink discoloration of the skin over joints (“frost erythema”). There may be black-brown spots on the gastric mucosa, (“Wischnewsky spots”), thought to result from terminal vasodilation of submucosal vessels. The kidneys may be pale with microscopic subnuclear vacuolization of the tubular epithelium (the “Armanni-Ebstein” lesion). Acute hemorrhagic pancreatitis has also been described. However, these findings require a period of survival to develop—many cases, particularly if the decedent succumbs quickly, show no findings at all. The diagnosis of hypothermia therefore relies heavily on scene investigation. “Paradoxical undressing” (demonstrated in this case), refers to the phenomenon of a terminally hypothermic person taking off their clothes. This is caused by a feeling of warmth resulting from failure of vasoconstriction in the skin, and contributed by altered mentation.

Those at greatest risk are people spending extended time outdoors, including the homeless and outdoor recreationalists. The elderly and very young have a lower ability to centrally regulate body temperature. Children’s increased body surface area also leads to more rapid heat loss. People who are intoxicated with alcohol or drugs may not sense the cold or lack judgment to seek shelter. Alcohol also acts as a vasodilator, impairing vasoconstrictive adaptation to cold.

As the weather cools down, be mindful of how easily hypothermia can develop. Temperatures can be above freezing, yet those who are vulnerable are still at risk of hypothermia. Prepare yourself well for any snowy excursions, and keep an eye on those in your community who may not be able to seek shelter.

*Stomach mucosa showing spots of black or dark brown discoloration*
*known as Wischnewsky spots. These are not specific to hypothermia and may just be an indicator of physiologic stress.*

*Bright pink discoloration over the knees, or “frost erythema”.*

*Pallor of the renal cortices corresponds to the microscopic “Armanni-Ebstein” lesion. This isn’t specific to hypothermia and can be seen in ketoacidosis from any cause.*

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

Triaging Times

As a clinical instructor and lead cytologist at my institution, I like to remind our newer cytologists and cytology students of the importance of being prepared for FNA biopsies so they develop good habits or best practices as they become more experienced. This level of preparation helps to create a culture of ongoing learning and improvement, which is necessary for the laboratory. In my experience, I’ve met some cytologists who prefer to go into a case blind, with the mindset that knowing the patient’s clinical history in advance muddies their knowledge, skills, and abilities, limiting their mindset by excluding the possibility of other diagnoses. While diving into the unknown might seem exciting, it is also a hindrance and could result in errors, especially when the clinical history helps us triage the patient’s sample. For example, knowing that the patient has a history of lymphoma or that the presentation state includes bulky lymphadenopathy prompts us to collect additional needle passes to send for flow cytometry analysis. Another concern is not knowing whether the patient has a history of breast, gastric, or esophageal cancer, and consequently processing the specimen routinely, which may result in an extended cold ischemic time. This delay in fixation along with insufficient formalin fixation can yield false negatives on ER/PR IHC in breast cancers and HER2 FISH in breast, gastric, and esophageal cancers, which could restrict the use of hormone therapies, such as tamoxifen and aromatase inhibitors for hormone receptor-positive (HR+) cancers, or trastuzumab for HER2+ cancers. I cannot overemphasize the importance of familiarizing yourself with clinical history and communicating case specifics while you act as a mediator between clinician and pathologist.

Whether the clinical history impacts the pre-analytical phase, such as specimen collection (limiting cold ischemic time or collecting additional needle passes for ancillary studies) or the analytical phase, as such processing (formalin fixation) and diagnosis (selecting an appropriate immunoprofile), we must remain vigilant and proactive in laboratory medicine. In this case, knowing the patient’s clinical history was of the utmost significance as it helped to reduce the number of immunostains and ancillary studies necessary to make the diagnosis. Using morphologic criteria in tandem with the patient’s clinical history narrowed the differential diagnoses to just two possible types of cancer, presented below.

A 59 year old male patient presented to the emergency room after an automobile accident. On imaging, the X-ray and CT scan identified a left humerus mass and fracture, and bloodwork was performed. His medical record was sparse and uneventful with no recent visits or encounters. To build a more comprehensive wellness profile and prepare for surgery, he was also offered a one-time screening for Hepatitis C, as an adult who was born between 1945 and 1965.

The left humerus mass was biopsied via CT-scan guidance and two passes were obtained. The Diff-Quik stained smears demonstrate large polygonal cells, some with abundant, granular cytoplasm and some isolated cells with naked nuclei. Vessels also appear to traverse some of the cell groups.

Images 1-2: Bone, Humerus, Left, CT-guided FNA. Diff-Quik-stained smears.

The Pap-stained smears also demonstrate polygonal cells with granular cytoplasm, nuclei with coarse chromatin, and prominent nucleoli. An interesting feature frequently identified in this case is the intranuclear inclusions, and in hindsight, a focus on these may have further reduced the number of immunostains performed.

Images 3-5: Bone, Humerus, Left, CT-guided FNA. Pap-stained smears.

The H&E-stained cell block sections show trabeculae with endothelial wrapping around the cell cords. While renal cell carcinoma was listed as a differential diagnosis due to its telltale oncocytic cytoplasm and vascularity, hepatocellular carcinoma was favored.

Images 6-7: Bone, Humerus, Left, CT-guided FNA. H&E sections (6: 100x, 7: 400x).

Immunostains were performed using proper positive and negative controls on the cell block sections, and the tumor cells show positive staining for Arginase, cam5.2, and Hepar1, while negative staining for CK7 and PAX8 (not shown).

Images 8-10: Bone, Humerus, Left, CT-guided FNA. Cell block section immunohistochemistry. 8: Arginase-positive; 9: cam5.2-positive; 10: Hepar1-positive.

Fortunately, before ordering immunostains, both our cytologist and pathologist working on the case peered into the patient’s medical record and noticed that he had recent bloodwork which demonstrated a positive Hepatitis C screening. This diagnosis was as recent as the identification of his humerus mass. Had it not been for his car accident, I can’t imagine how long he would have gone undiagnosed for both hepatitis and metastatic hepatocellular carcinoma. Incidental findings save lives, folks.

Granted, in settings of unknown primaries with widespread metastatic disease, such as carcinomatosis, an extensive workup is almost always inevitable. Narrowing down possible etiology based on information such as gender, age, and environmental or occupational exposure can help, but that doesn’t always yield a definitive answer as time- or cost-effectively as possible. In this case, that one clue of untreated Hepatitis C was all the cytopathology team needed. A rarity, sure, but as we are asked to do more personalized tests with less material, think of the patient’s specimen as a puzzle and keep your eye out for a clue both under the microscope and behind the computer. You never know what you might find that reduces errors and unnecessary testing while efficiently leading to a definitive diagnosis.

Microbiology Case Study: Severely Immunocompromised Female with Respiratory Failure

Case History

A 50 year old female with a complex medical history consisting of lymphoma, diabetes mellitus (type II), sarcoidosis, congestive heart failure, chronic renal failure (stage 3), and pancytopenia presented to the emergency department with shortness of breath, cough, fever. She was found to be positive for SARS-CoV-2 and was transferred to the ICU due to hypoxic respiratory failure. She was treated for sepsis and respiratory failure, but her status continued to decline. The patient had multiple admissions due to COVID-19 in the past, received remdesivir and was on corticosteroid therapy due to the interstitial lung disease from last year. Initial evaluation included complete blood count which revealed anemia (hemoglobin=8.7 mg/dl), leukocytosis (WBC = 21,900/mcl), lymphopenia (910/mcl) and thrombocytopenia (Plt = 27000/mcl). The patient was treated with broad antibiotics and additional steroids. Additional tests revealed hyperproteinemia and hypoalbuminemia. Chest x-ray showed worsening infiltrates in lungs and chest CT scan revealed left apical hydropneumothorax, loculated left pleural effusion, pneumomediastinum, and chest wall subcutaneous emphysema. Lung biopsy revealed necrosis. Histopathology examination revealed broad, branching hyphae with sporulation in lung tissue biopsy and bronchoalveolar lavage. Respiratory cultures of lung biopsy and BAL grew rapidly and lactophenol cotton blue tape preps showed broad hyphae with round sporangium and rhizoids between the stolons. The patient was diagnosed with mucormycosis, infection with Rhizomucor, and was treated with Amphotericin B. Surgical debridement of the tissue was not possible due to her declining condition. She passed away after 5 days.

Figure 1. H&E stain of the lung biopsy (top, left) and Papanicolaou stain of bronchoalveolar lavage (top, right) revealed broad, ribbon-like, right-angle branching hyphae (visible in lung biopsy) with sporulation (credits to Dr. Elham Arbzadeh, George Washington University School of Medicine and Health Sciences). Rapid growth was observed from the respiratory cultures of the tissue biopsy by day 2 (bottom, left) where lactophenol cotton blue tape preps showed broad hyphae with sporangium (bottom, right) and intermodal rhizoids (not shown in this image).

Discussion

The term mucormycoses refers to infections caused by the Zygomycetes which is further separated into Mucorales and Entomophthorales. Some of the members of Mucorales are Rhizopus spp., Mucor spp., Lichtheimia (Absidia) spp., Syncephalastrum spp., and Rhizomucor spp.^1,2 These organisms live in soil, dung, and vegetative matter. Infection is usually acquired by inhalation/ingestion of their spores or direct inoculation and contamination of wounds. The mold can invade the walls of the blood vessels causing angioinvasion and often results in dissemination of mycotic thrombi and development of systemic infection. Zygomycetes are most commonly known for causing rhinocerebral, pulmonary, cutaneous, and disseminated disease. Infections with Zygomycetes most commonly occur as opportunistic infections in immunocompromised hosts. Risk factors include diabetes, those with acidosis, neutropenia, and sustained immunosuppression such as after transplantation.

Zygomycetes grow very fast (within 48 to 72 hrs.) and is often called a “lid lifter”. The colonies have a wooly mycelium and can be described as cotton candy-like. Lactophenol tape preps of the mold would reveal broad hyphae, aseptate or pauciseptate, ribbon-like hyphae with irregular width. At the tip of the sporangiophore, there is a sack-like structure called a sporangia with contains all the spores. Fungal elements and hyphae seen on tissue biopsies from patients with mucormycosis typically have near right angle branching (usually >40^o) broad, non-septate hyphae. In contrary, those with aspergillosis show acute angle branching (usually <45^o) with narrow, septate hyphae.³

Genus-level identification can be achieved by microscopic morphology. Rhizomucor is an intermediate between Rhizopus and Mucor. Rhizoids found in Rhizomucor are few in number and are located on stolons, between the sporangiophores, as opposed to Rhizopus where the rhizoids are often seen directly at the nodes and Mucor which does not produce rhizoids. Sporangia (40-80 µm in diameter) are brown in color and round in shape. Apophysis is absent, which allows for differentiation from Lichtheimia (Absidia) where apophysis can be seen.⁴ The genus Rhizomucor includes three species: Rhizomucor pusillus, Rhizomucor miehei, and Rhizomucor tauricus.⁵

Treatment of mucormycosis consists of antifungal and surgical therapy. Amphotericin B is the most commonly used antifungal agent. Liposomal amphotericin B has also been successfully used in some cases with zygomycosis due to Rhizomucor.⁶ Early diagnosis and treatment are crucial and mortality rate is high.⁷ Of note, Zygomycetes are intrinsically resistant to voriconazole.

References

Rippon J W. Medical mycology. The pathogenic fungi and the pathogenic actinomycetes. Philadelphia, Pa: Saunders; 1974. Mucormycosis; pp. 430–447.
Scholer H J, Müller E. Beziehungen zwischen biochemischer Leistung und Morphologie bei Pilzen aus der Familie der Mucoraceen. Pathol Microbiol. 1966;29:730–741.
Mohindra S., Mohindra S., Gupta, R., Bakshi, J., Gupta, S. K. Rhinocerebral mucormycosis: the disease spectrum in 27 patients. Mycoses. doi: 10.1111/j.1439-0507.2007.01364.x.
de Hoog, G. S., J. Guarro, J. Gene, and M. J. Figueras. 2000. Atlas of Clinical Fungi, 2nd ed, vol. 1. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands)
Schipper M A A. On the genera Rhizomucor and Parasitella. Stud Mycol. 1978;17:53–71.
Bjorkholm, M., G. Runarsson, F. Celsing, M. Kalin, B. Petrini, and P. Engervall. 2001. Liposomal amphotericin B and surgery in the successful treatment of invasive pulmonary mucormycosis in a patient with acute T- lymphoblastic leukemia. Scand J Infec Dis. 33:316-319.
Ribes, J. A., C. L. Vanover-Sams, and D. J. Baker. 2000. Zygomycetes in human disease. Clin Microbiol Rev. 13:236-301.

-Maryam Mehdipour Dalivand, MD is a Pathology Resident (PGY-1) at The George Washington University Hospital. She is pursuing AP/CP training.

-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.

What’s NOT New in Cancer Care?

In June of 2017 just at the start of the annual American Society of Clinical Oncology (ASCO) meeting in Chicago, Illinois, there were at least 7 new FDA approvals for immuno-oncology agents targeting PD-L1 in cancer. At that time (2017), there were 2030 potential agents targeting 265 different targets across cancer including the modalities of t-cell targeted and other immunomodulators, cell therapy, cancer vaccines, oncolytic viruses, and CD3-targeted bispecific antibodies. Just three years later (2020), prior to the COVID-19 pandemic, this landscape had increased to 4720 potential agents targeting 504 targets across the same spectrum. That represents a 233% growth in these agents. Although only a fraction of these is “approved” (i.e., FDA approved and in use in patients clinically), many these agents are in clinical trials that require patient recruitment using pathology and other testing data. What does this mean for pathologists and laboratory professionals? Depending on the tumor being targeted and the target, there may or may not be a specific laboratory test that needs to be performed which may be routine, like histology parameters or immunohistochemistry, or may require advanced methods, like unique antibodies/clones, specific quantification methods, or molecular testing. The range of testing is not even unique to a specific therapy—for example, pembrolizumab uses staining for PD-L1, MSI, or no testing at all depending on tumor type. For the sub-specialized pathologist that focuses on one or two organs only, mastering the rapid pace and required diagnostic-therapeutic pairings is still a challenge. Imagine what it is like to be a general surgical pathologist in a community setting serving a community cancer center. Moreover, the diagnosis of a specific tumor is often completely disconnected for any biomarkers that may be indicated at the time of collection or several months later depending on therapeutic outcomes. This poses a range of problems in logistics and processing that are still being worked out at the individual system level. Still, the plethora of new treatments for cancer patients is very exciting.

In 2017, the largest group of targets (which was heterogenous) were tumor associated antigens (TAA) which are molecules that are not normally found in the human body produced by tumor cells as the result of changes to cellular processes. Whether it is hybrid proteins, glycosylation, or phosphorylation products, etc., these unique antigens held amazing promise as something we could target and destroy without fear of hurting normal human cells. However, the bulk of these approaches were for tumor vaccines (>90%) in 2017, dropping to 58% in 2020 (and from a total of 265 to only 198). To date, however, only a handful of cancer vaccines have been fully approved including sipuluecel-T for metastatic prostate and T-VEC for advance melanoma. This example category creates a complex set of challenges for pathologists and laboratory professionals. What data is needed about a patient or their tumor before a vaccine can be used? Does it require special studies that are not easily available or are costly? After vaccination, what follow-up tissue or blood studies are needed to follow the patient? Who dictates which tests are required before treatment: industry or medicine? But the more important challenge is: When do we, as the laboratory, pull the trigger to develop and disseminate such information and on-board new tests? Certainly, we are not going to look at Phase I trials and start taking about needs for future diagnostics. But by Phase III (where there is still a high dropout rate before full FDA approval) the number of potential agents and tests may still be daunting. If we wait until approval, now we are behind because our clinical colleagues will start immediately wanting to use the therapy. Tumor vaccines are an interesting category because we assume, for the most part, that there is likely only a diagnostic role needed. But then consider targets like CD-19, PD-L1, PD-1, CD3, Her2, CTLA-4, CD20, MUC1, CD22 and so on which are very familiar to our laboratory family because we often have already a test for these markers.

But is it the correct clone?

Do we have to score or interpret it differently?

When the agent is for cell therapy (the largest growth area of therapy development with 294% growth alone), what role does the transfusion medicine team play in administering or monitoring the patient?

As with the prior example, at what point do we, as a specialty of diagnosticians, dig into the forthcoming clinical trial results to plan? If our colleagues are in academic centers and are part of the clinical trials, they often are aware of and are administering the very tests that determine trial entrance. But if one reads just a few clinical trials of these agents, you may find that the inclusion criteria require a large battery of tests; however, on the other end when it is clinical ready for prime time, only one biomarker may be needed. Such a clustered landscape of information poses frustrating challenges for the clinical team and laboratory team in trying to find the way forward to get patients the life-saving therapies that are quickly arriving.

There is no question that the collision of targeted therapeutics and evolving diagnostics (i.e., precision cancer medicine) has demonstrated phenomenal growth with ever increasing benefits for patients. Affordability and access to these therapeutics aside*, studies continue to be completed and published including combinations therapies and hybrid therapies which show incredible promise. At ASCO 2022, the results of the DESTINY-Breast04 Phase III trial showed that trastuzumab deruxtecan (HER2-directed antibody and topoisomerase inhibitor conjugate) show a 49% reduction in the risk of disease progression or death versus physician’s choice of chemotherapy for patients with HER2-low metastatic breast cancer. That finding should be read a few times to make sure that the impact of this statement is very clear for pathologists and the laboratory. Previously, how we report HER2 (0, 1+, 2+, 3+) was complicated and often required FISH for questionable cases to look directly for HER2 amplification. This new category of patients requires reporting accurately 1+ or 2+ (FISH negative) disease, as it has incredible implications for patients. This news follows the recent new indications for CDK inhibitors in breast cancer related to Ki-67 mitotic score. Just when we thought breast cancer was straightforward, there is more to know and, more importantly, more time and tedium and standardization needed to report it for each patient. And, of course, early triple-negative breast cancer can also be treated with checkpoint inhibitors after PD-L1 testing is performed…but that’s literally old news as the data was release in 2020 at the start of the pandemic.

Outside of therapeutics, diagnostics are evolving quite rapidly with the COVID-19-induced ability to use digital pathology more readily creating a super-highway for artificial intelligence products to be validated for clinical use. PaigeAI has two such products (one for prostate and the second for breast lymph node evaluation released March of 2022) and many others are sure to follow. In parallel, screening, imaging, and surgery have also had advancements that continue to improve patient care and outcomes. So, it seems that everything feels new in cancer but is that the case?

The bulk of tumors diagnosed in the US (and elsewhere) are done with simply H&E staining (up to 75%) with another 20% being further confirmed by a few IHC tests (bringing the total up to 95%). This is not new and, most importantly, is the standard of care for the time being that we use to classify tumors. That classification has dictated, to some degree, the correct NCCN or other cancer protocol that oncologists used to treat patients. At some point, however, sufficient data on the bulk of all tumor types will likely point precision medicine treatments at all cancers. At that point, will a tissue biopsy be necessary with full histology or will a fine needle aspiration with molecular testing dictate the care? The credible assumption is that standard histology and IHC will remain in practice for the foreseeable future because so much billing, accreditation, and compliance is tied closely to them. But we CAN envision a “histology-free” oncopathology approach that matches patients to treatments with a panel of biomarkers. Sounds amazing but also stressful from the point of view of your typical anatomic pathologist.

*But the final thought on this, and perhaps the most important, is cost. Much like the domestic energy market is facing a dwindling pool of customers who agree to pay more and more for “traditional power” while their neighbors pump excessive kilowatts into the grid with their solar panels and windmills enjoying essentially “free power”, progress in cancer screening, detection, and treatment should be dwindling the pool of potential patients and increasing the costs to deliver care to the remainder. However, data and trends suggest that cancer is increasing globally. Why, if we are spending so much money and development on cancer care? Poverty and access. Cancer care is both expensive (in the US) and relatively expensive (in LMICs) with a focus on a small group of patients (0.55% of a population per year develop cancer). Projections of populations who need certain therapeutics are calculated using payer pools and markets that are existing and reliable. That does not include the bulk of LMICs. So, when we consider the cost of the PD-L1 checkpoint inhibitor class per year per patient is upwards of $125,000 USD, how can we even consider that an option for impoverished patients living off $1 USD per day? But if we don’t sort that out and treat these patients, we are assuming that persons who are impoverished are less valuable than persons who can afford expensive care. That evil logic, however, doesn’t hold true because even individuals in the US often become destitute or lose the bulk of their fiscal well-being when they must pay for cancer care—a situation that simply does not occur in countries with socialized medicine and/or universal healthcare.

Cancer care is rapidly evolving and the new tools and therapies available are incredible and miraculous for many patient types who would have faced a death sentence even 10 years ago. But with this amazing progress, we cannot ethically let people with limited resources succumb to these diseases over something so trivial as money. To do so poses harm and sets us up for failure as a species. It is for these reasons that ASCP engages in global health outreach. We are excited to have recently launched the Access To Oncology Medicines (ATOM) program with UICC and more than 2 dozen partners which will rapidly bring high-quality generic cancer therapeutics to low- and middle-income countries. In parallel with the St. Jude/WHO efforts on pediatric cancer globally, we will deliver quality cancer diagnosis and treatment to all patients everywhere.

If you want to learn more about PD-L1 testing and/or overcoming barriers to I-O in persons of color, new education from ASCP is available at no cost at https://www.ascp.org/content/learning/immuno-oncology/.

You can also check out our free educational resources on HER2-low breast cancer and Ki-67 testing in breast cancer at https://www.ascp.org/content/learning/breast-cancer.

Special thanks this month the Kellie Beumer (instructional design) and Melissa Kelly (monitoring and evaluation) from the ASCP medical education grants team for their thoughtful inputs into this piece.

References

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

Microbiology Case Study: How to “Pin” a Diagnosis

Case History

A 7 year old female presented to the emergency department with left sided abdominal pain and a temperature of 103 degrees Fahrenheit. Labs drawn showed mild leukocytosis with a CT scan suggestive of acute appendicitis. The patient underwent uncomplicated appendectomy with no complication. Gross examination of the appendix revealed an unremarkable, non-perforated serosa and a fecalith within the lumen. Representative tissue sections submitted for microscopic analysis per grossing policy. The findings below led to the submission of the entire appendix to be evaluated.

Figure 1. Low power image of an appendix demonstrating mild acute inflammation, lymphoid hyperplasia and congestion.

Figure 2. High power image, Cross-section of an adult female E. vermicularis from the same specimen shown in Figure 1. Adherent to the appendiceal surface. Note the presence of the alae (blue arrow), and the presence of almond shaped eggs (red arrow).

Discussion

The nematode Enterobius vermicularis, widely known as the human pinworm, is one of the most common parasitic worm infections today in the United States, infecting approximately 40 million people. The patient population is often children who are infected via fecal-oral transmission, with autoinfection being common. Humans are the only known host of this nematode. Once E. vermicularis embryonated oocytes are ingested, the larvae hatch and inhabit the gastrointestinal system. At night, the larvae migrate down to the anus, lay their eggs, and the cycle recurs.

The clinical presentation can be asymptomatic or can present with perianal pruritus at night, which can be explained via the life cycle of the parasite as stated above. The method of choice for diagnosing E. Vermicularis is microscopic examination of the eggs via cellulose tape slide test. A piece of scotch tape collects the eggs near the perianal area of the patient, which is then used for analysis and identification of the eggs. Microscopically, E. Vermicularis can be identified by the spines or ‘alas’ on the surface with oval shaped, thick capsuled oocytes within, seen in figure 2. To improve the sensitivity of the scotch tape test, it is best to do this test in the early morning, when there is an increased chance of sampling the eggs.

Rarely, is this worm associated with any severe symptoms but patients can present with abdominal pain, suggesting intestinal obstruction, extra intestinal manifestations like vulvovaginitis, or appendicitis. The relationship between E. Vermicularis and appendicitis is up for debate as to whether there is a causative relationship or if it is an incidental finding seen within appendicitis. Regardless of the relationship, once a diagnosis of Enterobius vermicularis is made, treatment with an anthelmintic needs to be given to the patient, such as Albendazole or Pyrantel Pamoate. In addition, treatment for everyone in the household needs to be considered in confirmed cases of infection.

Routine surgical specimens, such as appendices, can perhaps be overlooked once acute inflammation is noted. It is important to be able to identify organisms, such as pinworms, on such specimens to get the patient the appropriate treatment.

References

-Alexandra Medeiros, MD, is a first year anatomic and clinical pathology resident at Medical College of Georgia at Augusta University. Her academic interests include Forensic pathology, and surgical pathology.

-Hasan Samra, MD, is the Director of Clinical Microbiology at Augusta University and an Assistant Professor at the Medical College of Georgia.