Where have all the Techs Gone?

Electronic media is replete with articles and editorials of employers lamenting the shortage of workers. Signs offering hiring bonuses hang outside of restaurants, stores, and other retail outlets all across the country.

The inability to find workers has forced employers to take another look at their business model and reevaluate whether the model is still viable in its current form. The power balance in the employer/ employee dynamic has shifted. Employers accustomed to having their choice of applicants now find themselves scrambling to find workers.

No schools, No students

The healthcare industry, including the medical laboratory, is not exempt from the shortage despite healthcare experts and administrators knowing that the trending laboratory employee shortage was inevitable years ago.

Laboratory school administrators and managers have been sounding the alarm about the lack of community college and university medical technology program applications. Many academic medical technology programs are shuttered due to a lack of students.  The decrease in the number of students going into the laboratory field and the normal attrition rate of older workers retiring or moving on to higher-paying occupations has led to a high vacancy rate and a loss of expertise.

Burnout

The pandemic has added more pressure on a cohort of employees experiencing the stress of a new and unknown danger. These allied health professionals were (and are) the front-line response to a disease threatening everyone, regardless of economic or social demographics. Lab worker burnout has become a documented phenomenon

We call them heroes, but in reality, these are the same people working every day (pandemic or not), serving patients and delivering quality test results. Labs across the nation are filled with these everyday people. But just like everyone, laboratory workers have families, feelings, and needs they are trying to meet while being asked to give a little more. Many have little left to give and are now leaving the field to pursue other less stressful occupations or to simply enjoy the life they have worked so hard to build.

Start recruiting early

How can healthcare organizations stem the tide of those choosing to leave the lab and simultaneously attract young fresh minds to the unglamorous and less financially rewarding but necessary field of laboratory testing?

Presentations to elementary school children are a great way to introduce the next generation to the laboratory field. What child doesn’t like looking into a microscope to see their own red and white blood cells? Roadshows put on in junior high and high schools are a great way to kindle interest in healthcare just when students are beginning to ponder the question of what they want as a career.

Educational Aid

The cost of college continues to rise. Scholarships are often garnered by high-performing “A” students. But there is a pool of “B” students that could also benefit from financial assistance and would be just as welcomed into clinical laboratories. Broadening and diversifying the qualifications to receive a scholarship and financial aid could conceivably add to the pool of potential laboratory workers. Another unique idea is to allow laboratory workers’ dependents access to unused employee educational benefits.

Wellness in the Lab

Resources should also be dedicated to retaining technicians and technologists who are considering leaving the laboratory field.  The level of compensation is meaningful, but studies have shown that employees often leave the job for more esoteric reasons. Reducing stress, supporting a culture of wellness, inclusiveness, and belonging can differentiate one workplace from another. The theme of workplace wellness was extensively discussed at this year’s ASCP 2021 annual meeting in Boston.

The Need is Real

The pandemic has highlighted the importance of the laboratory to the health of the nation. The medical laboratory should use this moment in the spotlight to advocate for more resources and emphasize the necessity for more laboratory programs and students to meet the future testing needs of the nation.

Of course, many lab managers are wondering what to do today to stem the slow leak of personnel. Providing mental health support and financial incentives do work to keep these knowledgeable workers in the lab. Managers realize that laboratory science is a demanding high acuity job with little or no margin for error. To maintain quality, the healthcare industry will need to change its perceptions about the laboratory and address the lack of technicians and technologists with the same interest and retention resources given to nurses and doctors.

-Darryl Elzie, PsyD, MHA, MT(ASCP), CQA(ASQ), has been an ASCP Medical Technologist for over 30 years and has been performing CAP inspections for 15+ years. Dr. Elzie provides laboratory quality oversight for four hospitals, one ambulatory care center, and supports laboratory quality initiatives throughout the Sentara Healthcare system.

Disruption in Cancer Care: Good or Bad … What’s Next?

The concept of disruption often has negative connotations. Everyone on the planet can understand the phrase, “COVID-19 has disrupted our lives” without explanation. Although this disruption has been global, the disruption and ensuing impact this has had on non-COVID-19 related healthcare and, specifically, oncology, have been dramatic.

Surgeries, chemotherapy and other medical treatments were canceled or delayed by months, and volumes of testing across the cancer landscape dropped to minimums. Existing infrastructure furthered the deployment of telehealth consultations and, eventually, clinics were reopened; however, there is no question that many people with cancer face being diagnosed at a more advanced stage of disease, with worse outcomes.

On 25-26 October, the World Cancer Leaders’ Summit, organized by the Union for International Cancer Control and hosted by the American Society for Clinical Pathology, brought together more than 600 leaders from some 100 countries. One of the major topics of discussion was, “What do we do for oncology after COVID-19?”

In addition to examining heart-wrenching data on disruptions to cancer services, there were also positive discussions about what we have learned from this pandemic, how we have adapted, and what novel approaches we should keep that could create optimal, more efficient, or more impactful cancer care.

The positive side of disruption

When applied to innovative technologies or ways of thinking, “disruption” can be positive, particularly when we consider the many advancements happening so quickly with treatments, including immunotherapies like check-point inhibitors, mRNA cancer vaccines, CAR-T therapy, epigenetic therapies, that the different members of the cancer community are often running to catch up.

Some of these advances are simply operational efficiency (i.e., getting more output from the system by improving the inputs and the usage) while many are transformative innovations (i.e., immunotherapy for lung cancer and melanoma). And some advances are considered “disruptive” because they are not just a new way of doing something better but allow an entirely new approach that previously wasn’t available and that radically improves prevention, diagnosis, treatment or supportive care.

A disruptive revolution in cancer detection

In oncology, a true disruptive innovation is taking place with universal cancer screening (UCS) or multi-cancer early detection (MCED). The earlier a cancer is detected and the patient can start treatment, the higher the chance of survival. The current paradigm for cancer care is suspicion of cancer leads to diagnosis, which leads to treatment. Suspicion rests in either the results of a screening test or when a person shows symptoms, and diagnosis involves a biopsy that must be analyzed.

Primary care doctors and not just oncologists will be able to use UCS and MCED testing platforms. Tests will be performed on a timescale (e.g. annually, every five years) relevant to the person’s age, medical and family history as well as the type of cancer being detected for, rather than wait for a patient to present with symptoms. Furthermore, these platforms will be able to detect 20 to 50 or more cancers from a single sample and for myriad cancer stages, including precursor or pre-invasive cancer, and there is no need for a separate diagnostics phase: the result itself would dictate a treatment because the UCS/MCED platforms not only detect the cancer but can, in theory, give an origin and medical response parameters.

Whereas the current paradigm involves primary care, oncology, surgery, radiology, pathology, nursing, etc., this new paradigm would only involve primary care and an insurance provider.

Innovating, Creating and Breaking Down Barriers

The transition from traditional oncology to such novel platforms – as with all disruptive technologies – will not be smooth as we are talking about entire businesses and careers connected to traditional oncology possibly become obsolete. People with cancer, however, are expected to have shorter, more efficient journeys, likely with better outcomes and at a lower cost.

In LMICs, where oncology care systems are not nearly as developed as in HICs and where governments, unlike the US, are generally assumed or expected to pay for cancer services, UCS/MCED will require fewer dollars and provide better results than investing in the infrastructure required to create traditional cancer care systems. If this theoretical framework (UCS/MCED for cancer) does demonstrate the value in promises, it would set the stage for similar paradigms in other non-communicable diseases for which infrastructure and resources in LMICs are often lacking.

UCS/MCED was a hot topic at the WCLS. The leaders that were involved in the meeting sit on either side of a fence with regards to this innovation. There are those that support this technology’s development as quickly as possible, anticipating better patient outcomes, more efficient systems, less healthcare spending and more revenue. There are also opponents to this innovation, who throw up barriers resulting from fear of losses (revenue, employment, testing volume, referral networks, etc.).

The barriers they present, however, are important only if they are true barriers and not just perceived barriers. Why? True barriers are likely to require the engagement of the traditional oncology system to overcome; yet the act of overcoming those barriers may herald the disruptive innovation they fear. When an existing system must participate in its own creative destruction, can such a disruptive innovation take place?

No doubt the participants of the WCLS will continue to ask this question and let’s hope they find some answers for the sake of our patients.

milner-small


-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: A Young Adult in Septic Shock

A 23 year old female with a previous medical history of endocarditis, hepatitis C, IV drug use, and aortic insufficiency status post emergent aortic valve replacement, presented to the ER in septic shock. After one week of hospitalization, she left against medical advice, and did not complete her prescribed course of antimicrobials.

One month later, she returned to the ER with tachypnea, lactic acidosis, and altered mental status, secondary to septic shock and she was admitted to the ICU. She was started on broad spectrum antibiotics based on the cultures from her previous hospitalization. Within one day, blood cultures from her central line were positive for growth of Serratia marcescens. Echocardiogram demonstrated prosthetic valve endocarditis with severe aortic regurgitation. Previous imaging had shown scattered septic emboli throughout her viscera, extremities, and now, MRI/MRA revealed cerebral lesions as well.

Ten and twelve days into her current hospitalization, blood and heart valve tissue cultures (respectively) were both positive for growth of the below-pictured organism. What is this causative organism?

Image 1. Central line blood culture.
Image 2. Heart valve tissue culture.

MALDI-ToF-MS identified the yeast from the blood culture and heart valve as Trichosporon asahii. It is a yeast-like basidiomycete. It is commonly found in soil, but is also a normal colonizer of mucous membranes of the GI and respiratory epithelium, and skin. It may also infect hair shafts and is the causative agent in “white piedra”. It is involved in several opportunistic infections in the immunosuppressed. Of all Trichosporon species, T. asahii is the most common cause of disseminated infection, especially in those with hematologic malignancies (leukemia, multiple myeloma, aplastic anemia, lymphoma), solid tumors, AIDS, and solid tumors. In immunocompetent patients, Trichosporon may cause infections including endophthalmitis following cataract surgery, endocarditis, following prosthetic heart valve replacement (as seen in this patient), and peritonitis in IV drug abusers or those receiving continuous ambulatory peritoneal dialysis (CAPD).

Trichosporon colonies are powdery, cream-colored, and with age, may develop surface wrinkles. On cornmeal Tween 80, yeast can either grow alone or in short chains. True and pseudohyphae may be seen. Barrel-shaped arthroconidia are typically present. Variable growth is seen on media containing cycloheximide. It may also cause Cryptococcal antigen agglutination tests to be falsely positive.

Diagnosis is typically via blood culture.

Combination therapy with amphotericin B and an -azole drugs seems to be the most successful treatment option.

Resources

  1. Brandt, ME, Lockhart, SR. Recent developments with Candida and other opportunistic yeasts. Curr Fungal Infect Rep. 6(3); 170-177. 2012.
  2. Dimorphic Systemic Mycoses | Mycology | University of Adelaide Accessed 10/22/21.
  3. Love, G. Mycology Benchtop Reference Guide. College of American Pathologists. P20. 2013.
  4. Maves, RC. Trichosporon Infections. Emedicine.medscape.com. Updated Feb 12, 2018. Accessed 10/18/21.
  5. Procop, GW, Church, DL, Hall, GS et al. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. 7th Edition. P 1366-1369. Wolter’s Kluwer Health. 2017.
  6. Ramos, JM, Cuenca-Estrella, M, Gutierrez, F, et al. Clinical case of endocarditis due to Trichosporon inkin and antifungal susceptibility profile of the organism. J Clin Microbiology. 42(5):2341-4. 2004.
  7. Trichosporon | Mycology | University of Adelaide Accessed 10/22/21.

-Jenny Pfeiffer, MD is a 1st year Anatomic and Clinical Pathology resident at the University of Vermont Medical Center.

-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.

Microbiology Case Study: A 62 Year Old Male with Altered Mental Status

Case Description

A 62 year old male with unknown past medical history was dropped off at the emergency department by EMS after being found altered with concern for IV drug use. On presentation he was febrile to 104.5o F, tachycardic, and although he was initially responsive, his mental status deteriorated. Labs were drawn and broad-spectrum antibiotic coverage with vancomycin, cefepime, and metronidazole was initiated in the ED. He then had a tonic-clonic seizure event and was given intravenous levetiracetam. A CT brain showed a right inferior temporal lobe lesion, initially interpreted as likely glioblastoma multiforme, causing subfalcine and uncal herniation. MRI revealed a ring-enhancing mass measuring 3 cm x 3 cm x 3 cm in the right temporal lobe with significant surrounding edema. CT of the temporal bones also revealed right mastoiditis (Figures 1 and 2).

Figure 1. Coronal T1 post-contrast MRI demonstrating the ring-enhancing mass in the right temporal lobe (arrow).
Figure 2. CT of temporal bones with IV contrast demonstrating opacification of the right mastoid air cells and abnormal soft tissue within the epitympanum (arrow).

Neurosurgery evacuated 17 mL of fluid from the mass and a ventricular drain was placed. Gram stain of the evacuated fluid identified many white blood cells and few gram-positive cocci in pairs, chains and clusters (Figure 3). Postoperatively, the patient was mechanically ventilated and medicines were used to support his blood pressure in the ICU. Broad-spectrum antibiotics were continued for CNS penetration and activity against possible oral flora.

Figure 3. Representative Gram stain of the pus drained from the abscess. Multiple couplets of lancet-shaped gram positive organisms identified. Slight halo around the bacteria suggests the presence of a capsule.

An aerobic culture of drained contents from the brain ultimately grew characteristic alpha-hemolytic colonies with central umbilication (Figure 4) which were subsequently identified by MALDI-TOF and optochin disk as Streptococcus pneumoniae. Admission blood cultures also grew Streptococcus pneumoniae with characteristic “bullet-” or “lancet-shaped” gram-positive cocci in pairs on Gram stain. Fungal and acid-fast bacillus cultures had no growth. Following susceptibility testing; antibiotic coverage was narrowed to IV Penicillin G.

Figure 4: Representative, archival image of a blood agar plate with alpha-hemolytic, centrally umbilicated colonies and optochin susceptibility (P disk).

The patient remained unresponsive and required continued intensive medical support. Although blood cultures were sterilized, he continued to have fevers and persistent leukocytosis. Gram stain of ventricular drainage re-demonstrated gram positive diplococci. The patient was transitioned to comfort care and expired on day 5 of hospitalization, the cause of death was sepsis.

Discussion

This case of a brain abscess demonstrates an unusual intracranial complication of Streptococcus pneumoniae. S. pneumoniae (or pneumococcus) is a commensal of the upper respiratory tract (URT) and important opportunistic pathogen. Up to 65% of children and less than 10% of adults are colonized by S. pneumoniae. Dissemination of S. pneumoniae beyond its niche in the nasal mucosa leads to a spectrum of disease including lobar pneumonia, meningitis, sepsis, sinus infections and middle ear infections.1 Local dissemination of S. pneumoniae to the central nervous system (CNS) is the most common intracranial complication of otitis media and mastoiditis. These patients can present with fulminant “otogenic” meningitis. About a third of these cases require myringotomy or mastoidectomy.2 Focal parenchymal brain infection by pneumococcus, however, is uncommon.

This patient presented with signs of mass effect due to a large temporal lobe abscess warranting emergent neurosurgery. Broadly, focal parenchymal brain infections arise either by hematogenous dissemination of organisms or contiguous spread from an adjacent infection. The age, immune status, and any underlying disease present in the patient help predict the pathogen. Brain imaging is also helpful. Hematogenous spread, usually from endocarditis, tends to produce multiple lesions at the grey-white matter junction,3 while direct seeding causes solitary lesions.4,5 In this older patient with a relatively intact immune system and a possible history of intravenous drug use, hematogenous spread of bacteria was considered. However, a large single lesion in the temporal lobe with a plausible adjacent nidus (opacified mastoid air cells) is most consistent with contiguous spread.

A wide range of organisms should be considered when evaluating brain abscesses, though S. pneumoniae is a relatively rare culprit. A meta-analysis of 9,699 patients with brain abscesses found that S. pneumoniae was isolated from only 2.4%.6 The most common organisms were streptococci of the viridans group (34%) and Staphylococcus spp., most commonly S. aureus (18%). Even among patients with otogenic intracranial abscesses, S. pneumoniae is rarely implicated. Interestingly, the pathogen most frequently isolated from otogenic brain abscesses is Proteus mirabilis. 7,8

Once S. pneumoniae was identified, susceptibility testing was required to rule out acquired resistance to beta lactam and cephalosporin antibiotics, which is mediated by altered penicillin-binding proteins (PBPs).9 A more stringent susceptibility minimal inhibitory concentration (MIC) breakpoint applies to S. pneumoniae meningitis than other infections to account for drug distribution into the CNS.10 The hospital antibiogram reports that 97% of S. pneumoniae isolates are susceptible to Penicillin at MICs acceptable for treating non-meningitis infection but only 53% are susceptible at MICs for meningitis. Furthermore, 3.3% of all strains reported in the United States between 2001 and 2005 were also significantly resistant to ceftriaxone.11 This patient was covered by broad spectrum antibiotics until susceptibility testing demonstrated sensitivity to both penicillin and ceftriaxone.

References

1          Weiser, J. N., Ferreira, D. M. & Paton, J. C. Streptococcus pneumoniae: transmission, colonization and invasion. Nat Rev Microbiol 16, 355-367, doi:10.1038/s41579-018-0001-8 (2018).

2          Kaplan, D. M., Gluck, O., Kraus, M., Slovik, Y. & Juwad, H. Acute bacterial meningitis caused by acute otitis media in adults: A series of 12 patients. Ear Nose Throat J 96, 20-28 (2017).

3          Bakshi, R. et al. Cranial magnetic resonance imaging findings in bacterial endocarditis: the neuroimaging spectrum of septic brain embolization demonstrated in twelve patients. J Neuroimaging 9, 78-84, doi:10.1111/jon19999278 (1999).

4          Brouwer, M. C., Tunkel, A. R., McKhann, G. M., 2nd & van de Beek, D. Brain abscess. N Engl J Med 371, 447-456, doi:10.1056/NEJMra1301635 (2014).

5          Miller, J. M. et al. A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2018 Update by the Infectious Diseases Society of America and the American Society for Microbiology. Clin Infect Dis 67, e1-e94, doi:10.1093/cid/ciy381 (2018).

6          Brouwer, M. C., Coutinho, J. M. & van de Beek, D. Clinical characteristics and outcome of brain abscess: systematic review and meta-analysis. Neurology 82, 806-813, doi:10.1212/WNL.0000000000000172 (2014).

7          Duarte, M. J. et al. Otogenic brain abscesses: A systematic review. Laryngoscope Investig Otolaryngol 3, 198-208, doi:10.1002/lio2.150 (2018).

8          Kangsanarak, J., Fooanant, S., Ruckphaopunt, K., Navacharoen, N. & Teotrakul, S. Extracranial and intracranial complications of suppurative otitis media. Report of 102 cases. J Laryngol Otol 107, 999-1004, doi:10.1017/s0022215100125095 (1993).

9          Chen, L. F., Chopra, T. & Kaye, K. S. Pathogens resistant to antibacterial agents. Infect Dis Clin North Am 23, 817-845, vii, doi:10.1016/j.idc.2009.06.002 (2009).

10        Weinstein, M. P., Klugman, K. P. & Jones, R. N. Rationale for revised penicillin susceptibility breakpoints versus Streptococcus pneumoniae: coping with antimicrobial susceptibility in an era of resistance. Clin Infect Dis 48, 1596-1600, doi:10.1086/598975 (2009).

11        Sahm, D. F. et al. Tracking resistance among bacterial respiratory tract pathogens: summary of findings of the TRUST Surveillance Initiative, 2001-2005. Postgrad Med 120, 8-15, doi:10.3810/pgm.2008.09.suppl52.279 (2008).

Miles Black, Ph.D. is a fourth-year medical student in the Medical Scientist Training Program at UT Southwestern Medical Center. His background is in enzyme biochemistry and Legionella pathogenesis.

Denver Niles, MD is the Medical Microbiology fellow at UT Southwestern Medical Center. Prior to his Medical Microbiology fellowship, he completed pediatric infectious disease training at Baylor College of Medicine/Texas Children’s Hospital.

Dominick Cavuoti, D.O. is a professor of Pathology at UT Southwestern Medical Center who specializes in Medical Microbiology, ID Pathology and Cytology.

-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.

Microbiology Case Study: An Elderly Male Presents with Chest Tightness

Case History

An elderly male with a complex past medical history presented to the Emergency Department with the primary complaint of chest tightness for 2 days. He denied symptoms of diaphoresis, nausea, shortness of breath, palpitations, light-headedness, productive cough, dyspnea, chest pain, fevers, chills, or hemoptysis. He had no known sick contacts or recent travel. A computer tomography (CT) scan of the thorax showed a right hilar mass (Image 1). He underwent a bronchoscopy and right hilar transbronchial needle aspiration (TBNA) and bronchoalveolar lavage (BAL) were collected. The pathology report indicated abnormal lymphocytic proliferation, concerning for a mature small B-cell lymphoproliferative disorder.

Image 1. CT scan of the thorax showing the right hilar mass.

The BAL was submitted for acid-fast bacteria (AFB) culture, Gram stain, aerobic bacterial culture, and fungal culture. The AFB culture, Gram stain, and bacterial culture were all negative. However, 3 tan-yellow creamy colonies of a yeast grew on the sabouraud dextrose agar (SAB) plate in fungal culture after 7 days (Image 2). An India ink stain was performed (Image 3). MALDI-TOF confirmed the identification as Cryptococcus neoformans.

Image 2. Fungal growth on the SAB plate observed after 7 days.
Image 3. India ink staining of the fungus.

Discussion

Cryptococcus neoformans is an encapsulated pathogenic yeast, which is typically associated with bird droppings and contaminated soil.1,2 In immunocompromised patients, it can lead to severe opportunistic infections such as meningitis or disseminated disease. C. neoformans can cause life-threatening fungal infections in these patients, especially those with T-cell mediated immunodeficiency.3,4 The three main virulence factors include the complex capsule, melanin production, and ability to grow at human body temperature.5,6 Signs of pulmonary infection include cough, production of mucoid sputum, pleuritic chest pain, low-grade fever, dyspnea, weight loss, and malaise.

Fungal culture is one of the primary methods of Cryptococcus identification. Upon microscopic examination, Cryptococcus appears as a single bud and a narrow neck between parent and daughter cell and measures 4 – 10 uM.7 It has a fragile cell wall and a polysaccharide capsule that can vary from a wide halo to a nearly undetectable zone around the cells. Colonies can exhibit a wide range of color (i.e. cream, tan, pink, or yellow) and typically grow within one week of inoculation.8,9 India ink smear is a rapid method that allows direct visualization cryptococcal capsule, but is infrequently used now. Certain non-specific histological stains (including Periodic Acid-Schiff and May-Grünwald-Giemsa) can be used to detect fungi directly in fixed specimens. Fontana-Masson is a silver stain used for detecting melanin and has a high sensitivity for cryptococcosis.10  Other useful stains include hematoxylin-eosin, which reveals the clear halo, and mucicarmine and alcian blue, which target the polysaccharide capsule.11 Cryptococcal serology and cryptococcal antigen testing can be used for blood or CSF infections. Radiographic findings (especially in asymptomatic and immunocompetent patients) include patchy pneumonitis, granulomas (typically 2-7 cm), and miliary disease similar to tuberculosis.8 Treatment will vary depending on location of infection and host immune status. In some cases, pulmonary Cryptococcus may not be treated. Some clinical considerations include:

  • CSF chemistry parameters are normal
  • CSF culture, cryptococcal antigen, India ink preparation, and serology results are negative
  • Urine culture results are negative
  • Pulmonary lesion is small and stable/shrinking
  • No predisposing conditions for disseminated disease6

If treatment is required, fluconazole, itraconazole, or amphotericin B with or without flucytosine can be used depending on severity of infection.12

Cryptococcus gattii is another species of Cryptococcus. It differs from C. neoformans in that it typically infects both immunocompromised and immunocompetent patients. Canavine glycine bromothymol blue (CBG) agar can be used to differentiate C. gattii from C. neoformans: C. gattii is able to grow in the presence of canavine, turning the agar blue, while C. neoformans does not, leaving the media color unchanged.13,14

References

  1. Hagen, F., Khayhan, K., Theelen, B., Kolecka, A., Polacheck, I., Sionov, E., Falk, R., Parnmen, S., Lumbsch, H. T., and Boekhout, T. Recognition of seven species in the Cryptococcus gattii/Cryptococcus neoformans species complex. Fungal Genet Biol. 2015; 78: 16-48. 
  2. Lortholary, O., Nunez, H., Brauner, M. W., and Dromer, F. Pulmonary cryptococcosis. Semin Respir Crit Care Med. 2004; 25: 145–57.
  3. Lanternier, F., Cypowyj, S., Picard, C., Bustamante, J., Lortholary, O., Casanova, J. L., and Puel, A.  Primary immunodeficiencies underlying fungal infections.  Curr Opin Pediatr. 2013; 25: 736–47. 
  4. National Organization for Rare Disorders (NORD). Cryptococcosis. Available from: https://rarediseases.org/rare-diseases/cryptococcosis/ Last updated 2007; cited 2021 October 8.
  5. Idnurm, A., Bahn, Y.-S., Nielsen, K., Lin, X., Fraser, J. A., and Heitman, J. Deciphering the model pathogenic fungus Cryptococcus neoformans. Nat Rev Microbiol. 2005; 3(1): 753-64.
  6. Vandeputte, P., Ferrari, S., and Coste, A. T. Antifungal Resistance and New Strategies to Control Fungal Infections. Int J Microbiol. 2012: 713687.
  7. Guarner, J. and Brandt, M. E. Histopathologic Diagnosis of Fungal Infections in the 21st Century. Clin Microbiol Rev. 2011; 24(4): 247-80.
  8. Borman, A. M. and Johnson, E. M. (2020).  Candida, Cryptococcus, and Other Yeasts of Medical Importance. Manual of Clinical Microbiology, 12th Edition. Washington, DC: ASM Press. 2056-86.
  9. Coelho, C., Bocca, A. L., and Casadevall, A. The tools for virulence of Cryptococcus neoformans. Adv Appl Microbiol. 2014; 87: 1-41.
  10. Bishop, J. A., Nelson, A. M., Merz, W. G., Askin, F. B., and Riedel, S. Evaluation of the detection of melanin by the Fontana-Masson silver stain in tissue with a wide range of organisms including Cryptococcus. Hum Pathol. 2012; 43(6): 898-903.
  11. Guery, R., Lanternier, F., Pilmis, B., and Lortholary, O. Cryptococcus neoformans (Cryptococcosis). Antimicrobe. Available at: http://www.antimicrobe.org/new/f04.asp. Last updated: 2014; cited 2021 October 8.
  12. Perfect, J. R., Dismukes, W. E., Dromer, F., Goldman, D. L., Graybill, J. R., Hamill, R. J., Harrison, T. S., Larsen, R. A., Lortholary, O., Nguyen, M.-H., Pappas, P. G., Powderly, W. G., Singh, N., Sobel, J. D., and Sorrell, T. C. Clinical Practice Guidelines for the Management of Cryptococcal Disease: 2010 Update by the Infectious Diseases Society of America. Clinical Infectious Diseases. 2010; 50(3): 291-322.
  13. Larone, D. (2011). Medically Important Fungi. Washington, DC: ASM Press.
  14. Klein, K. R., Hall, L., Demi, S., Rysavy, J. M., Wohlfiel, S. L., and Wengenack, N. L. Identification of Cryptococcus gattii by use of L-canavanine glycine bromothymol blue medium and DNA sequencing. J Clin Microbiol. 2009; 47: 3669-72.

-Marika L. Forsythe, MD is a PGY1 Pathology Resident at University of Chicago (NorthShore). Her academic interests include molecular diagnostics and its growing importance in the field of pathology.

-Paige M.K. Larkin, PhD, D(ABMM), M(ASCP)CM is the Director of Molecular Microbiology and Associate Director of Clinical Microbiology at NorthShore University HealthSystem in Evanston, IL. Her interests include mycology, mycobacteriology, point-of-care testing, and molecular diagnostics, especially next generation sequencing.

E(cto)pic Metastasis

A 72 year old female originally presented with lung carcinoid and bilateral renal masses. The patient’s left kidney biopsy demonstrated ectopic thyroid parenchyma by an outside institution. Her thyroid function tests were unremarkable, she had no known previous head and neck radiation, and to the best of her knowledge, there was no family history of thyroid cancer. She underwent FDG PET imaging, which showed increased bilateral uptake in the neck (thyroid and lymph nodes), and an avid right posterior renal mass. Otherwise, her scan was relatively clear. Her left renal mass was resected and demonstrated thyroid parenchyma, but the differential diagnoses included thyroid heterotopia and metastatic well-differentiated thyroid carcinoma.

FNA and core biopsy were then obtained from the right upper quadrant of the kidney. The findings are depicted below.

Images 1-6: Kidney, Right, Fine Needle Aspiration. 1: Pap-stained smear; 2: DQ-stained smear; 3: H&E Cell Block section; 4: TTF-1+; 5: Thyroglobulin +; 6: CK7+.

The FNA was signed out as “Atypical thyroid tissue present.” Immunohistochemical stains demonstrated positive staining for CK7, vimentin (partial), TTF-1, thyroglobulin, and PAX-8 (partial), and negative staining for RCC, Napsin A, synaptophysin, and chromogranin. While these immunostains suggest thyroid-type tissue, morphology was most worrisome for metastatic thyroid carcinoma. The chromatin presented as hypochromatic and powdery, nuclear grooves and pseudoinclusions were present, and the nuclei were enlarged with irregular membranes. However, the scant material present precluded a definitive diagnosis.

Images 7-8: Kidney, Right, Core Biopsy. 7, H&E section 100X; 8, H&E section 400X.

The core biopsy suggested benign-appearing thyroid tissue similar to that seen in the left kidney, however, the surgical pathologist diagnosed the material as metastatic thyroid carcinoma.

A thyroid FNA was obtained from one of the patient’s multiple right-lobed thyroid nodules consistent with TI-RADS category 5 the next day. This was diagnosed as atypia of underdetermined significance due to scant cellularity.

Images 9-10: Thyroid, Right Lobe, Fine Needle Aspiration. 9: DQ-stained smear; 10: Pap-stained smear.

The right renal mass was resected after molecular profiling was performed on the left renal mass tissue. Mutation Detection by Next Generation Sequencing demonstrated a tumor mutation burden of 3.6Muts/Mb and identified mutations in the PRKDC, PTEN, and KRAS genes. Two kidney tumors were identified in the right kidney (one measuring 8.0 cm and the other 4.5 cm), both diagnosed as metastatic thyroid carcinoma with papillary features.

Images 11-12: Kidney, Right, Resection. 11, H&E section 40X; 12, H&E section 400X.

The thyroid was then resected, and pathologic findings were consistent with invasive follicular carcinoma with extensive angioinvasion to 4 or more vessels. While renal metastases are rare, the high affinity for angioinvasion makes the kidney a prime metastasis site due to its vascular-rich tissue. The patient was prescribed a low iodine diet and Thyrogen-stimulated radioiodine ablation to remove any remaining thyroid tissue or micrometastases and enhance the sensitivity of thyroglobulin as a tumor marker for surveillance purposes. While thyroid cancer (papillary and follicular types) is typically considered “the best cancer to have” due its slow growth and low-risk of widespread malignancy, it doesn’t mean that it won’t metastasize, even to a distant organ that you normally wouldn’t suspect. Great caution must be taken to ensure that lumps, bumps, and swallowing issues are addressed at annual physicals to catch a low-risk cancer before it has the opportunity to become an epic metastasis.

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

Pre-Analytic factors in NGS: Recognizing Contamination

Pre-analytic factors contribute to over 70% of laboratory errors. The most common examples include missing test request, wrong/ missing ID, contamination from collection site, hemolyzed/ clotted/ insufficient sample, wrong container or wrong transport conditions. 

In our NGS lab, pre-analytic factors rarely arise. Sometimes an incorrect slide was sent, so then the tumor and germline DNA don’t match. We do perform many rounds of PCR (total 30-35 cycles), so contamination of amplified PCR products could be a concern. Usually, our volume is low enough that contamination isn’t a significant issue and not one we have noticed.

Multiple mutations- A COVID-19 Hybrid?

In a batch of COVID-19 whole genome sequencing samples from early July, I noticed some interesting mutations. Mutations from the Alpha and Delta variants were showing up in the same specimen. At first I thought this could mean a hybrid virus. Another possibility was co-infection in the same patient. And lastly, the specimen could just be contaminated. The best way to resolve this issue is to check the “phase” of the viral variant sequencing reads. The phase refers to the single pieces of DNA that are read by the sequencer. They are normally short (75-150bp in length), but if you can see whether the variants occur on the same read or only on different reads it rules out the possibility of a hybrid virus.

Image 1. Hybrid (or chimeric) cat.

Determining the difference between a hybrid or 2 viral genomes

The difficulty is finding a location within both the Alpha and Delta variants that have mutations close to each other. One region exists near a deletion at amino acid 144 (Alpha) and amino acids 157-158 (Delta).

Figure 1. Characteristic mutations of Alpha (top) and Delta (bottom) variants. The deletions (triangle sign) are located close to each other, so were evaluated for phase.
Figure 2. Integrated Genome View (IGV) display of mutations. 6 base pair deletion (bars next to 6) is characteristic of Delta while a 3 base pair deletion (bars next to 3) is characteristic of Alpha.

Tracing the source of the issue

As there were no overlaping reads with the 3 b.p. and 6 b.p. deletion, we concluded that this finding arose from 2 distinct viral genomes. As to whether the person was infected with both Alpha and Delta, we looked to the rest of our 96 well plate. A characteristic muation in Alpha spike protein is N501Y (it confers increased binding to ACE2R and increased infectivity). This mutation was found in several specimens of the Delta lineage, but in this case there was a much lower frequency of this variant compared to total reads. Also, several of the cases had high CT values to start with. Many of the contaminated samples also were near a variant that mapped strongly to Alpha and had a lower Ct value (CT=25). Mapping the location of the specmens to the plate showed close proximity to the authentic Alpha variant.

Figure 3. Red squares: PCR=B.1.1.7; others are PCR=B.1.1.7, but yellow highlighting indicates N501Y detected in WGS.

Luckily, before all WGS testing, we perform a targeted PCR, which I’ve mentioned in previous blog posts. This showed that only one sample (well F3) was B.1.1.7, and the rest were Delta variants. Thus we concluded we had experienced a case of contamination.

Since this time, we’ve had issues with the negative control having borderline positive levels of sequencing data that maps to Delta (now 100% of all cases and with low CT values). This is apparently a problem at multiple labs, but one that we are trying to address by looking at several root causes.

Pre-analytic concerns: Contamination sources

COVID-19 viral genome sequencing has found issues of contamination in several circumstances. We attribute this to a few reasons:

  • High viral load of some specimens (especially Delta)
  • Thin plastic covers that pop off easily, especially when taken out of the freezer
  • And a large volume of specimens being processed.

For an example of the plastic cover issue, you can see this picture below where the cold temperature of -80C causes the plastic film adhesive to come off quickly as it is removed from cold storage. It makes popping sounds and could aerosolize viral particles to other wells in the plate.

Image 2. 96 deep well plate of extracted RNA with a thin plastic cover that comes off as the plates is thawed.

We now use aluminum PCR plate covers that do not come off with freeze/ thaw transitions. Furthermore, we use a multi-channel pipette that pieces the cover to withdraw individual samples without exposing them to other wells.

We have also implemented bioinformatic QC metric cut-offs to determine where a cut-off for negative specimens should be. We decided on an ambiguity score <0.5. The results were consistent with previous findings where lineages could be assigned at a CT >30, but sometimes they failed as CT values increased. Negative controls were assigned a CT of 40 and all fell below the 0.5 cut-off. This has been a useful metric to be sure we are providing high quality results.

Concluding remarks

Pre-analytic factors impact every part of testing and as COVID-19 sequencing has shown, even the NGS lab tests are not immune to these challenges.

The targeted PCR test helped flag/ resolve several of the issues as they arose.

COVID-19 sequencing is still for research/ epidemiologic purposes and demonstrates the importance of rigorous clinical validations to mitigate issues such as carry-over.

References Lippi G, Chance JJ, Church S, Dazzi P, Fontana R, Giavarina D, et al. Preanalytical quality improvement: from dream to reality. Clin Chem Lab Med. 2011;49:1113–26.

-Jeff SoRelle, MD is Assistant Professor of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX working in the Next Generation Sequencing lab. His research interests include the genetics of allergy, COVID-19 variant sequencing, and lab medicine of transgender healthcare. Follow him on Twitter @Jeff_SoRelle.

Moving Beyond Data to Action

On October 6th, 2021, the Lancet Commission on Diagnostics launched the “Transforming access to diagnostics” commission report with a virtual program and release of several publications. One of the publications included a study led by Dr. Sue Horton on access to diagnostics using data from 14 countries, mostly in Africa, from 2004 to 2018 with single timepoint data used to evaluate the relationship of access to diagnosis with a variety of factors. The diagnostics that were evaluated did not include histopathology, crucial for the diagnosis of cancer; however, the study did show importantly that income and population density had demonstrable relationships with access to diagnostics at the primary care level. For hospital-based access, there was no relationship which led the authors to conclude, among many other and relevant points, that access to diagnostics in “primary health care is the diagnostic so-called last mile and particularly affects poor, rural, and marginali[z]ed communities globally; appropriate access is essential for equity and social justice.” In the Commission report, the authors describe a tiered system with three levels that countries should incorporate into a national laboratory strategy and suggest that the burden of affording this system should fall on the governments. Moreover, they demonstrate the extremely important data around use of global markets to show that while the top four countries supply nearly 50% of all diagnostics, those same four countries only supply 24% of pharmaceuticals. In the opening statements to the Lancet Commission launch, Dr. John Nkengasong espoused very strongly the importance of manufacture of diagnostics WITHIN LMICs as one much needed solution.

For example, I was assisting a colleague with access to immunohistochemistry antibodies for which they were currently paying $700 USD for one vial of CD20. I traced the manufacture back to the US supplier (where the antibody was produced) and attempted to buy a vial as a private citizen with a credit card and was surprised to see that I could do so for $220 USD. This is the exact same vial of CD20 antibody. Why was my colleague paying a 218% markup? When I inquired with the company of manufacture, they reported that they had existing contracts to supply 2nd, 3rd, and 4th party vendors that they could not violate (i.e., they could not sell directly to a purchaser on the continent of Africa). The local supplier charging the $700 USD suppled a very large number and breadth of medical supplies including other diagnostic tests and reagents. Those reagents were reasonably priced, and several were on sustained government contracts. However, the CD20 antibody was not. Why is that the case? Let’s assume you are a supplier of widgets and wobbles. Your demand for widgets is huge and you sell more than 100,000 widgets per month to 20 different consumers. For wobbles, one person orders one wobble once per year. Your widgets ship room temperature but your wobbles require a cold chain, lest they be destroyed. What would you do? You could choose not to sell wobbles. You could choose to charge a ridiculous price for wobbles so that the excess time, energy, and expense of getting one wobble to your consumer is worth the effort. But you would not sell the wobbles for a similar profit margin as your widgets. It just wouldn’t make business since. Now imagine that the wobbles are manufactured in a country other than your own and to get them, you buy them from a country supplier who buys them from a regional supplier. So, wobbles already come with additional markups. You do have a third choice which is to manufacture wobbles locally, cut out the middle people, and charge much less but still make more profit. This is a great model if wobbles can be easily manufactured; however, when wobbles require an enormous capital investment, is it worth it to sell a couple of wobbles a year? Of course not. This business-based example is one of the drivers for a $700 USD vial of CD20. If a local manufacturer, in country or in a neighboring country, could manufacture and sell, this reagent would be more affordable and feasible as an available diagnostic. Specifically, patients with lymphoma would have access to rituximab for CD20.

But note the Commissions finding that almost 50% of diagnostics are made in the top 4 countries. This means, naturally, that the pricing for these reagents and supplies will be based on that economy and/or GDP, not on the economy or GDP of every country down to the lowest on any given scale. Consider the Big Mac Index, which looks at buying power relative to the US dollar. The only African country used in the Big Mac Index is South Africa and it is third from the bottom. To be clearer, if you have 100 South African Rand, you could get about $6.69 USD if you exchanged it directly (ignoring fees). If you want to buy a Big Mac in the USA, the average consumer price is $5.65; however, in South Africa, it’s 33.50 Rand. Based on the Dollar:Rand exchange rate, we are paying only $2.24 USD in South Africa for the same sandwich that would cost us $5.65 in the US. So, the Rand is undervalued. Now, let’s look at our vial of CD20 (not revealing the country to protect identities). According to the current exchange rate, you get $4.34 USD for every 10,000 units of this countries currency. Based on this model, if the CD20 was being EVENLY exchanged with cash (as opposed to being undervalued or discounted as we saw with South Africa), it should cost 450,586 units of this country’s currency. Instead, it is costing them 1,612,053 units. If we assume that this country could/should achieve a Big Mac Index equivalent discounted of the CD20 as we see with the Big Mac itself in South Africa, it should cost them 307,057 units or $133 USD. The difference? The Big Mac is manufactured and locally distributed directly to the customer in South Africa. The CD20 is not. So, one step to achieving an equitable pricing structure in healthcare for LMICs, especially in Africa, needs either direct discounted by US- and European-based manufacturers—unlikely to occur because of fear of alternative market access—or these products need to be manufactured and supplied locally.

What I have trouble agreeing with completely and, in some cases, even it part, is the concept of all healthcare costs falling on the government of a population with the expectation that they deploy a “one size fits all” approach to any aspect of healthcare. When we consider the US and Europe (again, the top four producers of diagnostics), we find one as a largely private commercial system driven by government pricing for elder care and the other a socialist system with universal healthcare enhanced by private care. For both systems, there is a huge economic base which either drives capitalism across the system from raw materials to final product or an enormous tax base that can cover the bulk of the costs of the systems. As we move from these four down the GDP ladder to LMICs, we don’t see, despite that we would like to nicely categorize countries into clear groups, a solution that would work “globally” because major pieces of economic development are needed as pre-requisites for a capitalist open market or one payer system. Each country has a unique set of circumstances (e.g., history, genetic diversity, geography, natural resources, tourism, disease burden, language, population size, etc.) that cannot be reduced to simply a GDP value or Big Mac Index factor. Moreover, it is wholly within the realm of colonialism, which we supposedly abandoned 70 to 80 years ago, to think that we can propose a system for “all countries” that would even remotely approach the solving the problems of these countries. Although it is an excellent mental exercise to idealize a healthcare system as having something as simple as three tiers and trying to allocate what tools and resources are needed at each level to accommodate the population, the reality is that such a framework is only a starting point with a lot of work needed to fully realize what type of system would be best for a given country. Very small islands and small nations may have only one hospital to serve its entire population and insufficient patients of a given type to justify the expense of certain tools. Extremely large countries with large populations will need a myriad of systems with their own tiers that support patients based on location, socioeconomic status, language, etc. and these systems likely overlap in geography. And the expertise to best determine that system is the health and government leadership of that country, not an external set of non-specific instructions. The external set of instructions, however, are extremely important, as noted, as a starting point, but each country that identifies a gap in their diagnostics, for example, has to assess their specific situation. At the heart of this problem is the need to stop talking about the challenges of global healthcare and start (or continue) directly working on fixing them.

At ASCP, we approach our global outreach through assessment, gap identification, implementation planning, and execution (AGIE). Through that approach, we have deployed and/or support 19 sites in 15 countries with telepathology; however, in an additional 10 countries, we have active programs that have not yet reached a point of telepathology deployment. Had we said, from the beginning, “We are going to give everyone telepathology”, we would have wasted an enormous amount of time and money. By following an AGIE approach, we have navigated to the specific problems of each site with whom we collaborate and attempted to solve them. And we do so with more than 80 collaborative partners. The Lancet Commissions on Diagnostics most recent launch is an excellent first alert for those who have not been engaged in global health for the last 20 years that there are still major challenges and problems in global healthcare and diagnostics. Our hope is that funders, governments, industry, health system members, patients, and advocates will view this as a rallying cry to direct resources and energy to join those of us who have been engaged in this work to move the needle even further. Access to diagnostics for every patient everywhere. It is ASCP’s simple mantra, and we hope, together, we can achieve that goal.

References

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-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.

The Anatomy of Lab Safety Design: Handling a Flood

Most laboratories are designed with eyewash stations and at least one safety shower depending on the size of the department. The use of these safety showers is not common, but it does happen, and the staff needs to be prepared for such an event. That preparation not only involves testing and training on equipment use, but also in making sure the physical space is ready for a potential deluge of water that can pour down into the department for potentially up to fifteen minutes. Other flooding incidents may occur as well. A floor drain can back up, a water line connected to an analyzer might break, or water might even come through the ceiling from a pipe above the department. Being prepared and responding efficiently to these types of flooding events should be part of the overall lab safety program.

One reason safety specialists and some regulatory agencies require that items in the lab not be stored directly on the floor is so they will not be damaged in the event of a departmental flood. It is generally acceptable to store plastic items (waste bins, etc.) on the floor since they cannot be damaged by water. Cardboard, computer hard drives, and other like items should be stored on palettes or shelves. Securing electrical wires and raising multi-plug adaptors off the floor is also a best practice.

When designing or remodeling a laboratory, consider the possibility of floods when choosing the type of flooring to be installed. The best laboratory flooring is monolithic, like a sheet vinyl that has few seams. It should bend up to the walls to create a coved base that is integral with the floor. This design (recommended by the CDC and CLSI) keeps liquids from going under tiles or through walls which will create more problems (like mold) down the road.

Floor drains where safety showers exist are not required, and many labs have showers where there is no drain at all. Remember that in a typical situation where a shower would be used, hazardous chemicals are involved. Any hazardous waste that might go into the sanitary sewer should be routed through a neutralization station or into a hazardous waste collection tank. The ANSI requirements for a safety shower include the ability to deliver 20 gallons of water per minute for 15-20 minutes. That’s a total of 400 gallons. The requirements also state that the water pattern must be at least 20” in diameter and 60” above the floor. Therefore, a majority of the water will not even travel to the drain. It will go to the lowest point of the floor in the department. The bottom line is, if the safety shower must be used, a flood should be expected.

In order for the lab to be prepared for a flood emergency, materials should be on hand that will help contain large amounts of water. Those materials may include large volume spill kits with booms or dikes that are capable of holding water back. Staff should be trained how to use these materials as spill training is provided, and drills should be conducted so they can use the supplies comfortably. Make sure these spill materials are easily accessible and that signage clearly indicates where they are stored.

What does the physical anatomy of your lab look like? Is it designed for safety in the event of a hazardous material spill or exposure? Is the department set up to handle a sudden flood situation, and can staff identify the steps to take to respond efficiently and safely? Take a look around your lab today, and make any necessary corrections so that all will be ready should a laboratory flood occur for any reason.

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.

Hematology Case Study: Too Many Platelets?

Too many platelets? We know that low platelet counts can pose problems for hematology analyzers and that reporting accurate results is vital for good patient care. We learn and read a lot about thrombocytopenia and its various symptoms, causes, and treatments. But, what about thrombocytosis? What happens when there are too many platelets?

In my last blog I compared 2 cases of newly diagnosed CML. Lately I have seen so many new leukemia cases and myeloproliferative diseases that I have become fascinated with them. When I was in college and grad school (many moons ago), nomenclature, diagnoses and knowledge of these disorders were very different, so it’s been fun learning about them all over again!

Today’s case is of a 55 year old woman who was referred for a hematology consult because of a finding of increased RBC and platelet counts. White blood cells appeared normal with few reactive lymphocytes noted. The peripheral smear showed mild anisocytosis and dacrocytes. Platelets were markedly increased with large forms present. No giant platelets were noted. A bone marrow biopsy was ordered. Pre-Op diagnosis: Thrombocytosis.

Bone marrow results reported increased myeloid forms with full spectrum of maturation, erythroid elements normal in number with normoblastic maturation, and markedly increased megakaryocytes with numerous large hyperlobated forms. M:E ratio was increased. No iron was seen on iron stain. A reticulin stain showed mildly increased reticulum fibrosis (1+). Next generation sequencing studies demonstrated a JAK2 V617F mutation. BCR-ABL mutation was not detected. Diagnosis: Myeloproliferative neoplasm most consistent with Essential Thrombocythemia (ET).

Myeloproliferative neoplasms (MPN) are a group of disorders characterized by the over proliferation of WBCs, RBCs, or platelets. These can be separated into the Philadelphia chromosome (Ph) positive Chronic Myelogenous Leukemia (CML) and Ph negative neoplasms. The BCR-ABL oncogene is formed on the Ph and is responsible for the unregulated proliferation of cells seen in CML. At diagnosis over 90% of CML cases are BCR-ABL positive. (See Case Studies in Hematology: Presenting a double feature starring Chronic Myelogenous Leukemia). On the other hand, Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are the three classic Ph negative neoplasms.

Many ET patients have no symptoms at diagnosis but are found to have a high platelet count on a routine CBC. Diagnosis is based on ruling out other disease and testing for genetic mutations, which can be done from a peripheral blood sample or bone marrow. In addition to any blood tests, a bone marrow biopsy is typically recommended for differential diagnosis of MPNs. The most common mutation found in PV, ET or PMF, is in the JAK2 gene. The JAK2 V617F mutation is found in nearly all PV patients, and about 50-60% of ET and PMF patients.Othermutations found in the classic MPN group include CALR, the second most common genetic abnormality after JAK2 mutations,and MPL W515L.Normally, blood cells are only produced when the body has a need for the cells, but these genetic mutations turn a gene ‘on’, causing the unregulated production of the affected blood cell line. Until recently it was believed that a patient with PV, ET or PMF will have a mutation in only one of these genes. However, in 2018, a French group reported that CALR or MPL mutations may co-exist in a small percentage of patients with a low burden of JAK2 V617F mutation. (Accurso) Some patients are triple-negative for the JAK2MPL and CALR mutations and always have a poor prognosis.

The identification of a genetic marker in MPNs is valuable because a JAK2 mutation distinguishes PV from other disorders that may cause polycythemia. As well, a JAK2 or other mutation can distinguish ET from other causes of reactive thrombocytosis and PMF from secondary causes of myelofibrosis. In addition, most CML cases are diagnosed with a very high WBC, but occasionally patients with CML have a normal or only slightly elevated WBC with a high platelet count. Therefore, patients with suspected ET are also evaluated for CML with a test for the Philadelphia chromosome. Our patient was found to have a JAK2 V617F mutation, BCR-ABL negative and was diagnosed with ET.

ET was first recognized in the 1950’s and was termed a myeloproliferative disorder. At this time, it was not known what was causing the over proliferation of platelets. Theories were broad and ranged from ‘something environmental’ to ‘an internal defect’. Over the decades, it became more apparent that the myeloproliferative disorders were caused by internal defects in stem cells, and they were renamed MPN. In 2005, four separate research groups, using different methods, all identified the JAK2 V617F allele, which led to further understanding of PV, ET and MPN. The MPL mutation was discovered in 2006 and CALR mutations were discovered in 2013.

ET is a type of chronic leukemia and patients with ET generally have a normal life expectancy. Of the 3 BCR-ABL negative MPN, ET has the best prognosis. Treatment is often not needed, other than aspirin for prevention of blood clots. Patients are placed in risk factor groups based on risk of clots or bleeding. A patient <60 years with no JAK 2 mutation and no prior thrombosis is considered very low risk and would be simply observed or prescribed low dose aspirin. Patients <60, JAK2 V617F +, with no prior thrombosis have low risk and would be treated with aspirin, dosage dependent on any cardiac risk factors. Older patients over 60 with JAK2 wild type and no history of thrombosis may be treated with aspirin alone or with cytoreductive therapy. Lastly, the highest risk patients are those over 60, JAK2 V617F + or with prior thrombosis, and would be treated with cytoreductive therapy, such as hydroxyurea. With very high platelet counts, there is a risk of both blood clots and hemorrhage. Blood clots that develop in thrombocythemia can use up the body’s platelets and result in bleeding. For this reason, cytoreductive therapy such as hydroxyurea is recommended to reduce hemorrhage in high-risk patients with very high platelet counts over 1,000 x 103/ μL. Hydroxyurea can also be used as treatment in patients who have a mixed population of PV and ET. CALR mutated patients with ET tend to be young with a much lower thrombotic risk and do not generally require therapy. Aspirin in this group is considered overtreatment because CALR+ patients suffer more risk of bleeding with aspirin.

While there is some risk of a MPN transforming to another type, ET is the MPN least likely to transform or to progress to acute myeloid leukemia. ET also has a better prognosis than the other MPN. Even so, there is often not one clear cut entity. There can be overlap between the disorders, causing some difficulty in diagnosis and treatment decisions. For instance, a physician may have a patient, as our patient does, with a high RBC and Hgb, with thrombocytosis, and with a JAK2 mutation. Bone marrow biopsy may detect hyperlobated megakaryocytes which would indicate a diagnosis of ET; however, the physician may choose to monitor and possibly treat as PV due to the RBC counts and symptoms.

Many advances in the understanding of ET and molecular techniques for diagnosis have been made in the last 10 years. Unfortunately, many times, diagnosis is not made until after a thrombotic event. In addition, many patients with thrombocytosis are not referred for hematology consults in a timely fashion or until they too experience a thrombotic event. In 2016 WHO published a new diagnostic criterion for PV, ET and PMF. There is an effort amongst research and physician groups to ‘spread the news’ throughout the medical community to promote early detection of ET, minimize the risk of thrombotic events and improve prognosis.

References

  1. Accurso V, Santoro M, Mancuso S, et al. The Essential Thrombocythemia in 2020: What We Know and Where We Still Have to Dig Deep. Clin Med Insights Blood Disord. 2020;13:2634853520978210. Published 2020 Dec 28. doi:10.1177/2634853520978210
  2. Bose P, Verstovsek S. Updates in the management of polycythemia vera and essential thrombocythemia. Ther Adv Hematol. 2019;10:2040620719870052. Published 2019 Aug 30. doi:10.1177/2040620719870052
  3. Kilpivaara, O., Levine, R. JAK2 and MPL mutations in myeloproliferative neoplasms: discovery and science. Leukemia 22, 1813–1817 (2008). https://doi.org/10.1038/leu.2008.229
  4. Panjwani,Laura. Management of ET, PV Requires 2 Distinct Approaches. Special Reports, Hematologic Malignancies: Polycythemia Vera, Volume 3, Issue 3. September 28, 2016
  5. https://rarediseases.org/rare-diseases/essential-thrombocythemia/
  6. https://www.mpnconnect.com/pdf/who-diagnostic-criteria-mf-pv-et.pdf
Socha-small

-Becky Socha, MS, MLS(ASCP)CMBBCM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 40 years and has taught as an adjunct faculty member at Merrimack College, UMass Lowell and Stevenson University for over 20 years.  She has worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. She currently works at Mercy Medical Center in Baltimore, Md. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.