The Syncytial Variant: A High-Risk Subgroup Amongst the Traditionally Well-Behaved Classic Nodular Sclerosis Hodgkin Lymphomas

Lymphoid neoplasias are traditionally categorized as Hodgkin Lymphomas or Non-Hodgkin Lymphomas. Hodgkin Lymphomas are typically characterized by both expansive reactive lymphocytes and a paucity of the interspersed, malignant Reed-Sternberg (RS) Cell. These RS cells are large neoplastic B-Cell variants with ‘owl-eye’ nucleoli within multilobed nuclei. The background reactive lymphocytes are non-neoplastic cells drawn by secreted RS cytokines (IL-5, IL-6, IL-13, TNF, and GM-CSF), which often result in presenting B symptoms (fevers, chills, and night sweats). Histiocytes, granulocytes, and plasma cells are also commonly identified. Confirmatory immunohistochemistry staining for RS cells involves CD30 positivity, CD15 positivity, and negativity for CD20 and LCA (CD45) staining. The two major types of Hodgkin Lymphoma are recognized as Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL) and Classic Hodgkin Lymphoma (CHL). In NLPHL, B-Cell immunophenotype is generally preserved and is histologically recognized by a nodular predominance of small lymphocytes and RS cell variants, called lymphocyte predominant (LP) or popcorn cells (formerly called L&H cells for lymphocytic and/or histiocytic RS cell variants), which exhibit muted, lobed nuclei and smaller nucleoli. The Classic Hodgkin Lymphomas are categorized histologically as Nodular Sclerosis CHL (NSCHL), Lymphocyte Rich CHL (LRCHL), Mixed Cellularity CHL (MCCHL), and Lymphocyte Depleted CHL (LDCHL).

Nodular Sclerosis Classic Hodgkin Lymphoma (NSCHL) comprises over 70% of all CHL and is characterized by broad, fibroblast-poor collagen banding surrounding at least one nodule and by RS cells with lacunar morphology. The RS cells tend to have a larger amount of cytoplasm than those in other types of classic Hodgkin lymphoma. When fixed in formalin, this excess cytoplasm causes their membranes to retract so that the cells seem to be sitting in lacunae. Typically, EBV association is uncommon, CD30 is almost always expressed, CD15 is expressed in 75-85% of cases, and PAX5 is weakly positive. Most cases do not express T-Cell antigens (CD4, CD3, CD8), but when they do (~5% of cases), they can be difficult to distinguish from ALK-negative Anaplastic Large Cell Lymphomas (PAX5 positivity can help to rule out ALCL). True T-cell marker expression on RS cells has been seen (usually weak) and is associated with a poorer prognosis.

Two histologic variants of NSCHL have been proposed: a fibrocystic variant (FV) and a syncytial variant (SV). The FV contains abundant fibroblasts and histiocytes in the setting of difficultly identified RS cells. The SV describes the finding of RS cells in prominent, sheet-like aggregates forming cohesive lacunar nests in the center of nodules (Figure 1). The SV represents 5-16% of all cases of NSCHL (Ben-Yehuda-Salz et al. 1990), though some suspect it may be as high as 25% (Sethi, T., et al. 2017).

Advancements in chemoradiation therapies have improved the long-term survival of patients of with CHL. Today, more than 85% of patients with early stage CHL will be cured of the disease (Ansell, S., et al. 2014). The 5-year overall survival for early stage CHL is 95% and for advanced disease is 82%. Overall, the prognosis of NSCHL is better than that of other types of CHL.

Initial studies examining the prognostic significance of grading of NSCHL based on cytological and tumor microenvironment features showed an association of higher grade (based on the British National Lymphoma Investigation (BNLI) criteria (Bennett et al. 1981; MacLennan et al. 1989)) with a poorer prognosis (MacLennan et al. 1989; Wijlhuizen et al. 1989; Ferry et al. 1993). However, since the advent of combination chemotherapy and the relatively good prognosis of NSCHL today, the significance of such grading or further classification has been called into question. Importantly, grading based on proposed histological features has declined in the last two decades because such advances in therapy can actually obscure the differences seen in less-effectively treated patients. As such, grading is not currently deemed necessary for routine clinical purposes.

Despite the broadly recognized clinical favorability of NSCHL, slowly accumulating evidence of poor clinical outcomes associated with the syncytial NSCHL histological variant is only now beginning to declare itself with strong evidence. While BNLI grading may no longer be a useful clinical tool, variant typing may instead prove effective as both a prognostic and alternative treatment indicator.

Small case studies have reported aggressive disease in SV NSCHL since the first histologic case was described. Clinical behavior has included presenting with a considerable mediastinal mass, significant B symptoms, and advanced stage at diagnosis. Despite these case reports, it was not until recently that systematic analysis of progression free survival and complete therapy response among morphology and immunophenotype SV confirmed cases in patients with similar treatment regimens (combination doxorubicin, bleomycin, vinblastine, and dacarbazine, or ABVD) was conducted (Sethi, T., et al. 2017).

Sethi, T., et al evaluated 167 patients with NSCHL: 43 patients with SV were compared with 124 patients with typical NSCHL on patient characteristics, disease variables, treatment administered, and outcome at Vanderbilt University Medical Center between 1995 and 2014. The rate of complete treatment response was lower in the SV variant as compared with typical NSCHL with standard induction therapy, 74% versus 87% (p= 0.05). Patients with SV had a shorter progression free survival and experienced disease relapse. The median progression free survival for the entire cohort was 174.7 months. The median progression free survival in the SV group was 17.02 months which was significantly shorter compared with that of the typical NS group, which was not reached (p < 0.0001).

The BNLI criteria for the grading of NSCHL (Grades I-II) are based on the amount of sclerosis, the degree of nodular cellularity, and the number of atypical neoplastic cells. Most SV cases are grade II because of the number of atypical cells present. However, most grade II cases are not syncytial variants. Therefore, BNLI grade alone is not an accurate depiction of the clinicopathologic features of this disease. In the same way, it may also not be the best determinant of disease prognosis, therapeutic indication, or investigative classification.

Patients with Syncytial Variant Nodular Sclerosis Classic Hodgkin Lymphoma experience a lower than expected rate of complete therapeutic response with shorter progression-free than non-SV NSCHL treated with standard therapy. Syncytial Variant NSCHL should therefore be recognized as a high-risk subgroup within the otherwise traditionally docile NSCHL classification. It is time the SV finally be considered a true histopathologic variant in future trials involving novel agents to assess treatment response. While a majority of patients with SV NSCHL can likely be successfully salvaged with high-dose therapy and autologous stem cell transplantation, studies of novel agents such as conjugated antibodies or immunotherapeutic agents should be considered in these patients to improve complete response rates and to avoid the need for toxic salvage therapies.

References

  1. Sethi T., et al. Differences in outcome of patients with syncytial variant Hodgkin lymphoma compared with typical nodular sclerosis Hodgkin lymphoma. (2017) Ther. Adv. Hematol. 8(1):13-20.
  2. Granot, N., et al. Syncytial variant of nodular sclerosing Hodgkin lymphoma in children: A prognostic factor? (2018) J. Ped. Hem. & Onc. 35;1:33-36.
  3. Ansell, S. (2014) Hodgkin lymphoma: 2014 update on diagnosis, risk stratification, and management. Am J Hematol 89: 771–779.
  4. Bennett, M., Maclennan, K., Easterling, M., Vaughan Hudson, B., Jelliffe, A. and Vaughan Hudson, G. (1983) The prognostic significance of cellular subtypes in nodular sclerosing Hodgkin’s disease: an analysis of 271 non-laparotomised cases (BNLI report no. 22). Clin Radiol 34: 497–501.
  5. Ben-Yehuda-Salz, D., Ben-Yehuda, A., Polliack, A., Ron, N. and Okon, E. (1990) Syncytial variant of nodular sclerosing Hodgkin’s disease. A new clinicopathologic entity. Cancer 65: 1167–1172.
  6. Ferry, J., Linggood, R., Convery, K., Efird, J., Eliseo, R. and Harris, N. (1993) Hodgkin disease, nodular sclerosis type. Implications of histologic subclassification. Cancer 71: 457–463.
  7. Maclennan, K., Bennett, M., Tu, A., Hudson, B., Easterling, M., Hudson, G. et al. (1989) Relationship of histopathologic features to survival and relapse in nodular sclerosing Hodgkin’s disease. A study of 1659 patients. Cancer 64: 1686–1693.
  8. Wijlhuizen, T., Vrints, L., Jairam, R., Breed, W., Wijnen, J., Bosch, L. et al. (1989) Grades of nodular sclerosis (NSI-NSII) in Hodgkin’s disease. Are they of independent prognostic value? Cancer 63: 1150–1153.
  9. Strickler, J., Michie, S., Warnke, R. and Dorfman, R. (1986) The “syncytial variant” of nodular sclerosing Hodgkin’s disease. Am J Surg Pathol 10: 470–477.
  10. Van Spronsen, D., Vrints, L., Hofstra, G., Crommelin, M., Coebergh, J. and Breed, W. (1997) Disappearance of prognostic significance of histopathological grading of nodular sclerosing Hodgkin’s disease for unselected patients, 1972–92. Br J Haematol 96: 322–327.
  11. Hess, J., Bodis, S., Pinkus, G., Silver, B. and Mauch, P. (1994) Histopathologic grading of nodular sclerosis Hodgkin’s disease. Lack of prognostic significance in 254 surgically staged patients. Cancer 74: 708–714.
  12. Darabi, K., Tester, W., Daskal, I. and Cohn, J. (2015) Syncytial variant of nodular sclerosing Hodgkin’s disease. Blood 104: 4533–4533.

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-Austin McHenry is an M3 at Loyola University Stritch School of Medicine in Maywood, IL. Austin is past-president of the pathology intrest group SCOPE (Students Curious about Outrageous Pathology Experiences) and is a recent recipient of ASCP’s Academic Excellence and Achievement in Pathology Award. Follow Austin on Twitter @AustinMcHenry

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-Kamran M. Mirza, MD PhD is an Assistant Professor of Pathology and Medical Director of Molecular Pathology at Loyola University Medical Center. He was a top 5 honoree in ASCP’s Forty Under 40 2017. Follow Dr. Mirza on twitter @kmirza.

Ebola 2018

Approximately two years after Liberia, the hardest hit and last of the 6 countries to be affected in the largest Ebola outbreak since discovery of the disease in 1976, was declared Ebola-free, the virus has again reared its head. This time, its in the Democratic Republic of the Congo (DRC).

Timeline of the Outbreak:

  • May 3, 2018: a district in the Province of Equateur, DRC, reported 21 cases of undiagnosed illness with 17 deaths. Samples from 5 of these cases were sent to the Institute National Recherche Biomedicale in Kinshasa.
  • May 7: Ebola virus was confirmed by RT-PCR.
  • May 8, 2018: Ebola outbreak declared.
  • May 21: 628 contacts of confirmed or suspected cases listed.
  • May 25: 58 cases and 27 deaths.
  • June 1: the outbreak is contained in the Province of Equateur. This Province covers an area of 130442 km2 and has a population of 2,543,936. Equateur as 16 health zones and 284 health centers – this works out as 1 health center for every 9,000 people! The WHO warns that this outbreak has the potential to expand, and while at the moment there is no international spread, the Congo’s neighbors have been placed on alert. The WHO has distributed personal protective equipment, infrared thermometers, and rapid diagnostic tests to health centers in Equateur as well as neighboring countries.

The WHO considers laboratory diagnostics on of the pillars of the Ebola response. They recommend “strengthening diagnostic capabilities” as part of a strategic approach to the prevention, detection, and control of Ebola. In fact, laboratory diagnostics might be a key to how this epidemic plays out, versus the previous outbreak in West Africa wherein six African countries were affected and over 11,000 patients died. This time, there are rapid tests tests available ranging from lateral flow to molecular.

As part of the DRC’s National Laboratory Strategy developed in response to the outbreak, the GeneXpert confirmatory Ebola PCR test is being used a key sites in mobile laboratories. As of June 1, the WHO has deployed four mobile labs through out Equateur including the epicenter of the outbreak. Government Health Centers are equipped with rapid lateral flow tests: the ReEBOV Antigen Rapid Test released under Emergency Use Approval in 2015. According to WHO documents, this test has a sensitivity of 91% and specificity of 84.6%. Both positives and negatives should be confirmed with RT-PCR. The following is the guidance for the use of rapid tests:

Special settings where rapid antigen for Ebola may be beneficial:

  1. In the investigation of suspected Ebola outbreaks in remote settings where PCR tests are not immediately available. While awaiting confirmatory testing, action can be taken to: a) isolate test-positive patients, b) repeat daily testing on patients who initially tested negative but remain symptomatic, c) mobilize transport of samples for confirmatory testing and initiate outbreak-management procedures.
  2. In settings where the number of cases and suspects arriving for triage and care cannot be managed with the existing health staff and laboratory facilities.

Example situations where rapid antigen detection tests should NOT be used:

  • Individual case management – including for establishing definitive diagnosis or making therapeutic decisisions
  • Certification of Ebola virus-free status prior to medical care for other illnesses
  • Release of Ebola patients from Ebola Treatment Centers
  • Pooled blood samples for community-based testing
  • Testing blood before transfusion
  • Active case finding without confirmatory PCR
  • Any setting where action (quarantine, referral, care) based on results is not possible
  • Airport screening

So to summarize, currently in the Province of Equateur, suspected cases are tested by rapid test for initial triage, then samples are sent to the nearest lab for confirmation (positive or negative) by PCR. A suspected case cannot be released until there is a negative test by PCR. Suspected cases that initially negative by the rapid test are isolated from cases that are initially positive.

What about outside Equateur? I talked to Dr. Tim Rice, a friend and colleague serving as a missionary physician in Vanga, Congo. Vanga is the in Province of Bandundu, the northern neighbor of Equateur. While this province has not had a reported case of Ebola, they are getting ready. I asked him about their readiness plan and any laboratory capabilities they had. They have a rapid test: Ebola rapid lateral flow test from STADA Diagnostik (Germany). This assay detects the Ebola virus antigen VP 40 with a sensitivity of 92% and specificity of 98% (according to the package insert). Serum and throat swabs are acceptable specimens, although it is not clear which matrix was used to determine the performance characteristics. The package insert states that the performance characteristics are still being evaluated. Dr. Rice said they use the rapid test with patients with potential exposure and severely ill with fever.  Someone arriving from the Equatorial province with a fever, even if not severely ill, would be tested and isolated. They are to call the local health department for help in obtaining the correct confirmatory samples, properly storing the sample, alerting the regional and national leaders, and transporting the sample properly protected the 10 hours overland to Kinshasa for confirmatory PCR testing at the Institute National Recherche Biomedicale.

The response to the 2018 Ebola outbreak has been impressive and I sincerely hope that with the benefits of laboratory diagnostics and a vaccine, the world will be spared the devastation experienced in the previous outbreak.

 

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Sarah Riley, PhD, DABCC, is an Assistant Professor of Pediatrics and Pathology and Immunology at Washington University in St. Louis School of Medicine. She is passionate about bringing the lab out of the basement and into the forefront of global health.  

Microbiology Case Study: A 28 Year Old Female with Perirectal Abscess

Case History

A 28 year old female with a history of Ulcerative Colitis on humira and azathioprine presented with proctitis and a recent perirectal abscess. The patient reported a two week history of progressively worsening pain and swelling in the perianal region. In addition, she reported recent purulence excreted with bowel movements.  On physical exam, the patient was afebrile and negative for rash, oral lesions, joint pain, or abdominal pain. A perirectal abscess was identified and drained. Abscess culture was positive. Patient reported recently engaging in high-risk sexual behavior with multiple male sexual partners often without protection.

Lab Identification

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Image 1. Kidney-bean shaped gram negative cocci identified on Gram staining of perirectal abscess culture (100x oil immersion).  
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Image 2. Aerobic growth of flat, gray-white, and moist colonies from perirectal abscess on chocolate agar plate.  

Abscess culture grew kidney-bean shaped gram negative diplococci. Colonies on chocolate agar plate appeared medium sized, flat, gray-brown, and moist. The organism was oxidase positive and identified by MALDI to be Neisseria gonorrhoeae.

Discussion

Neisseria gonorrhoeae is a kidney-bean shaped gram negative diplococci for which humans are the only host. The organism causes gonorrhea, a common sexually transmitted disease, among young people between the ages of 15-24 years. Gonorrhea is spread by sexual contact or through the birth canal. The most common site of infection is the urogenital tract.2 Males commonly present with dysuria with penile discharge.2 Females commonly present asymptomatically or with symptoms such as mild vaginal mucopurulent discharge and severe pelvic pain2. In addition, gonorrhea can cause infections of the anus, conjunctiva, pharynx, ovary and uterus.2 In the neonate, the culprit organism can lead to ophthalmia neonatorum.2 Lastly, gonorrhea causes disseminated disease such as arthritis, endocarditis, meningitis, and skin lesions on extremities.2 CDC currently recommends treating gonorrhea with dual therapy, a single dose of 250 mg intramuscular ceftriaxone and 1g of oral azithromycin.1

Antibiotic resistance in gonorrhea is an increasing public health concern. The World Health Organization has a program that monitors the global antimicrobial resistance of gonorrhea.3 The data from 77 countries between 2009 and 2014 showed that 66% of reporting countries had encountered gonorrhea strains with either resistance or reduced susceptibility to ceftriaxone.3 81% of reporting countries had encountered gonorrhea strains resistant to azithromycin.3 Given these data, it is important to improve gonorrhea prevention and continue to monitor gonorrhea antibiotic resistance at both the national and global levels.

References

  1. Gonorrhea treatment and care. (2017, Oct 31st). Retrieved on March 1st, 2018 from https://www.cdc.gov/std/gonorrhea/treatment.htm
  2. Miller KE. Diagnosis and Treatment of Neisseria gonorrhoeae Am Fam Physician. 2006 May 15:73 (10): 1779-1784.
  3. Wi T, et al. Antimicrobial resistance in Neisseria gonororheae: Global surveillance and a call for international collaborative action. PLoS Med 14(7): e1002344.https://doi.org/10.1371/journal.pmed.1002344

 

-Ting Chen, MD is a 1st year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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-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.

History of Generation: Traditionalists

Traditionalists make up the smallest percentage involved in the current workforce, but they are the organizational historians as they know and remember the organization’s past and founding goals. Traditionalists are typically born between 1927 and 1945 and grew up during the Great Depression, which was from 1933 to 1938. After that, the second World War started and the U.S.A got involved after the attack on Pearl Harbor in 1941.

These years had a significant effect on this generation. Traditionalists are known to work collaboratively, know how to do more with less, and are task-oriented. They typically have a strong sense of what is right and wrong, which was fueled by the historical events in their childhood and early adulthood. They have a strong sense of patriotism and respect for authority figures.

This generation is also one of the first major innovators; they created space travel, vaccination programs, and the foundation for modern-day technological innovations. They were the driving force of the civil rights movement of the 50s and 60s and were also the ones that started moving to suburbs. Currently, the are serving on many Board of Directors, as Presidents of organizations or as executive leaders. They have generally moved up in the hierarchy of organizations that they have spent years working for. They are loyal employees who require little feedback from their managers.

Because this is the era of pre-feminism women, the majority of women raised children and only had a job before marriage as teachers, nurses, or secretaries. This generation is self-disciplined, cautious, and self-sacrificing.

 

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-Lotte Mulder earned her Master’s of Education from the Harvard Graduate School of Education in 2013, where she focused on Leadership and Group Development. She’s currently working toward a PhD in Organizational Leadership. At ASCP, Lotte designs and facilitates the ASCP Leadership Institute, an online leadership certificate program. She has also built ASCP’s first patient ambassador program, called Patient Champions, which leverages patient stories as they relate to the value of the lab.


 

This generation was born before 1945 and is the oldest generation in the American culture. However, not all of those born before 1945 are alike.  They either fought in WWII or were children through those war years.  The Traditionalist generation are really the first strong innovators and if they are still working they act as the historians of the organization because they have been there for a long time. They often serve on Board of Directors and are Presidents because of their organizational knowledge and expertise. They are typically very disciplined, consistent in their behavior and opinions, and are known for their loyalty.

The majority of Traditionalists are retirees and are the largest lobbyist group, which is the AARP.  If your parents or grandparents were of the Traditionalist Generation, you might have experienced a “waste not, want not” attitude with strong family values, conformity, and team players.

The Traditionalists are often referred to as the “Silent Generation.” This term came from the fact that during this era, the children were often expected to be seen and not heard.

As I pondered this generational topic, I found myself searching for an example of an “Active Working Traditionalist” that I could talk about because they might not have yet retired!  To my surprise I found myself thinking about my Uncle Tom.  This man has taken care of me and his family of five children with my Aunt Pat my whole life. He is a strong family man and then realized he is still working! Uncle Tom (he prefers to remain nameless) turned 83 year’s old this past April 16th.  He is still the principle owner of his own CPA firm and worked those long and hard CPA hours during this 2018 tax season.  As I mentioned early in this blog, all Traditionalists are not alike, and Uncle Tom never expected children to be “silent.”  He valued their opinions, and my Aunt Pat was both a stay home mom and a partner in their CPA firm.

Uncle Tom values the old-time morals of family first, safety, conservatism, patience and financial security.  I encourage you to look around for your Traditionalist at home, or maybe even in the workplace.  Let’s appreciate our Traditionalists while we still have the opportunity to learn from them!

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-Catherine Stakenas, MA, is the Senior Director of Organizational Leadership and Development and Performance Management at ASCP. She is certified in the use and interpretation of 28 self-assessment instruments and has designed and taught masters and doctoral level students.  

You Should Follow Troponins, Or They’ll Follow You

Hi everyone! Back with another piece about the life between the lab and medical school. This time, I’d like to take a minute to talk about some new and exciting developments in laboratory diagnostics happening right now: immunoassays for critical troponins are undergoing an evolution. Fourth generation testing is slowly developing into its fifth-generation upgrade. Labs across the country are starting to discuss the relatively new FDA approved fifth-gen cardiac troponin T assay which has been shown to be a high-sensitivity test. But what does this mean for labs? Specifically, what does it mean between the bench and the bedside? The hospital I’m currently on service at is rolling out the first beta-test of this assay in New York City right now, and as it turns out—it’s going to change a lot. Not only will the new understanding of cardiac enzyme reference ranges need a complete overhaul but tailoring appropriate clinical responses to those values will need to be looked at as well. I’m not a sales rep and this isn’t going to be an adventure in comparative statistical analytics, but I think it’s a great time to have a conversation early on about what these new generation assays could mean for us in the lab.

A Whole New World

When I was in graduate school, doing my MLS training we were taught the same cardiac enzyme assay history that was developed over the last 50 or so years. Early acute markers of inflammation relating to acute myocardial infarctions (AMIs) with respect to acute phase reactants AST, LDH, CRP, etc. As more technology advanced, specific biomarker analyses of individual detection of things like CK or myoglobin became useful. The WHO criteria for AMI then established (and re-established since the 1970s) the laboratory requirements for CK-MB and detectable levels of troponin to correlate with clinical findings. Further sensitivity and specificity developments, and clinical research like the GUSTO and APACE trials, showed us just how sensitive newer (then troponin T and I) cardiac assays could be. Not to mention, instead of rule-in/rule-out criteria, we had the development of risk stratification. And as instrumentation developed so did our testing—CK and LDH replaced with CK-MB and its isoforms, AST went the way of Myoglobin, and LD ratios became reliable troponins!

troponin-tab-1
Table 1. In each stage of acute coronary syndrome (ACS) various biomarkers are available and detectable in peripheral blood. The challenge has been to find the most reliable and time-sensitive cardiac enzyme(s) to reflect appropriate staging and risk stratification. Research is helpful, but clinical intervention required critical timing.

Ask most clinicians today about cardiac enzyme, cardiac injury studies, or other related markers and you’ll hear about CPK, CK-MB 1, 2, 3 and other isoenzymes, LDH electrophoresis, and of course troponins T and I. What’s more is that the reference ranges for most of these tests haven’t really changed much either. If I called you and said your patient in 706-W has a Troponin of <0.03 you might be relieved for now. If I said that same patient’s troponin was 0.560—we might have a different story unfold. But what if I told you your patient’s troponin was 13, and was trending down from 15? What do you do with that? What if I only called to report a troponin-DELTA which was 0.0? Was there an in-service you missed? Possibly. Sounds like your institution might have 5th-gen on board.

What was wrong with the old troponin?

In a word: nothing, really. This really isn’t about buy-in for a “better” test or a better detection method. This is about creating a dialogue about improving risk stratification for our patients with coronary disease. Let’s go back to Chicago, specifically the last hospital I worked in before starting medical school: Swedish Covenant Hospital. Having been through a few hospitals in my time, I can say you’d be hard pressed to find a more streamlined, albeit small community hospital, laboratory service. Running a full gamut of SIEMENS instrumentation and critical middleware-software, the management there ran a tight ship—which included critical troponins. We ran the TnI-Ultra assay on the ADVIA Centaur/XP platform. It was your standard three-part immunoassay sandwich test with a biotin-streptavidin antigen-antibody detection. It was fast, used little reagents, was relatively stable, had a great system of QC, and was calculably-flexible between heparinized and ETDA plasma samples. Two-point calibration kept it tight between (and this is from memory, loosely) 0.006 and 0.50 ng/mL, and I believe we called our critical values at 0.40 ng/mL. This was a good test, and it’s used in many labs today still. It’s got great stability and has room to interpret ACS risk stratification based on population data in each location. People understand those results, too. But exactly how much room between, let’s say 0.10 ng/mL and >0.50 ng/mL, is there to stratify that risk? Complex decision algorithms then become hybrids of institutional cardiology recommendations, American College of Cardiology (ACC) recommendations, emergency department input, and, of course, laboratory management recommendations.

Here at Bronx Care Hospital Center (BCHC), I spoke with a laboratory manager about rolling out this brand new high-sensivity troponin (hs-cTn), and we discussed their vaildation and policies. Along with this new project, the hospital has been a vocal part of American College of Cardiology ACC17 Acute and Stable Ischemic Heart Disease program addressing topics including marijuana use and ACS, stable angina risk stratification, NSTEMI sex differences in revascularization and outcomes, treating cocaine related ACS with beta blockers, and research Anticoagulation Therapy After Anterior Wall ST-Elevation Myocardial Infraction in Preventing LV Thrombi. So, it seems fitting that this is as good as any a pilot location for cardiology departments city-wide to watch and learn from the hs-cTn roll-out!

Tell me more about this new test…

This hs-cTn assay is an electrochemiluminescence immunoassay (ECLIA) that uses two monoclonal antibodies against human cardiac troponin T. At this institution, they are using this immune sandwich assay on a Roche Cobas E with a similar biotin-streptavidin coated microparticle complex as with the previous generation testing. This is a short test with similar reagent use and stability as before, and only for lithium heparinized samples. With a relatively quick turn-around in less than 10 minutes, the new hs-cTn offers critical information for clinical correlations on the fly from potential STEMI codes coming into the ED.

Without going into horrific details about validating data on old and new troponins across patient populations, there is something interesting to note here which came up in discussion with the lab manager: new reference ranges. Now, with testing sensitivity, cross-reactivity, ranges, and interfering substances, the ranges are no longer the decimal-place values reported that we’re all used to. The ranges we work with now at BCHC are cutoff between <12 ng/L for positives and >52 ng/L for critical values suggestive of acute coronary syndrome. The analytical measuring range is much larger now between 6-10,000 ng/L. With the adjustments for limits of detection and blanks on instrumentation, the specificities of these values are normalized on a larger index for reflecting differences in male and female cardiac enzyme activity to a common cutoff of that <12 ng/L value. But more so than just a value, a new part of trending troponins becomes much more important: the delta values. These are more acutely indicative of the cardiac necrosis and/or condition of other non-specific heart tissue damage en vivo occurring in patients. Taking all this into account, you now have a much wider and broader range of values to interpret and incorporate into your clinical decision making, which brought up a few questions when I spoke with lab staff as well as cardiologists.

So, what’s wrong with the new troponin?

Okay, that’s a fair question at this point. And my answer is still: probably nothing. Sorry to be so inconclusive, but it’s still early days. There is a lot of data to support moving toward newer generation hs-cTn testing since it has been available outside of FDA-approval in Europe before January 2017. Research done in the Department of Internal Medicine and Central Institute for Medical Laboratory Diagnostics at Innsbruck Medical University in Austria show that new troponins might not be that different (read: better) than their 4th generation counterparts, at least with regard to emergency room visits. T. Ploner, et. al, argue that diagnosing AMI in the ED doesn’t really benefit from the heightened sensitivity offered by the new Roche 5th gen assay (Figure 1, Figure 1). But, when they compared the detection of other cardiac disease including AMI, unstable angina, heart failure, arrythmias, pulmonary emboli, pulmonary disease, anemia, renal disease, and several other entities, the 5th generation assay could more readily detect changes early and provide clinicians with critical data quickly (Figure 1, Figure 2).

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Figure 1. Ploner et al. demonstrate here that there isn’t really any difference in the sensitivity of 4th generation vs. 5th generation troponins for detecting acute MI in the emergency room. However, there is a significant advantage in the detection of any cardiac disease, generally.
(Source: Ploner et al. (2017) High-Sensitivity cardiac tropnonin assay is not superior to its previous 4th cTnT assay generation for the diagnosis of acute MI in a real-world emergency department, link: http://spo.escardio.org/eslides/view.aspx?eevtid=48&fp=P4177)

Finally, I think a review paper from the American Journal of Medicine last year summarized it best. Coming from the Department of Cardiovascular Diseases and Department of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, MN, the authors discussed concern over the troubling over-sensitivity and potential pitfalls of reaching too far with hs-cTn testing. Their bottom line: collaborating on data and accuracy between cardiology, laboratory medicine, and emergency medicine, there is a great potential for this super sensitive test to provide clinicians with very useful data in the near future. We just have to process that data correctly. As always, results should be interpreted in conjunction with clinical presentation including medical history and laboratory data. But in the case of new and coming advances in critical care, there seem to be some common themes between my conversations here at BCHC and in what I read in the literature regarding how to ensure we move forward appropriately.

  1. Multidisciplinary educational efforts are critical. The ER, the lab, and the cardiology department at each institution utilizing 5th gen troponins need to understand the new ranges, the new triaging cutoffs, the clinical correlations for consulting the ICU/CCU, and how to understand the deltas for their patient populations.
  2. Create clear communications for your laboratory values. Will you normalize for gender or provide sex-specific confidence interval reporting? Will you provide tables for suggested value correlations with AMI/ACS protocols?
  3. Order sets and in-service training. You’d be surprised how much the nitty gritty details of lab draws and ordering appropriate tests/tubes could slow down your institution’s advancement.
troponin-fig-2
Figure 2. How the authors at Mayo Clinic establish the use of new hs-cTn assays in diagnosing and triaging potential AMI patients.
(Source: Sandoval, Jaffe (2017) Using High-Sensitivity Cardiac Troponin T for Acute Cardiac Care, The American Journal of Medicine (2017) 130, 1358–1365, doi:10.1016/j.amjmed.2017.07.033)

At the very end of the day, it’s up to the institution. Clinical centers have to follow their own guidelines for cardiac pathology. ACC/ESC/AHA guidelines and Universal MI definitions are for clinical correlation across locations, but a single roll-out of a fancy new test can’t make a better ER. It really does take communication, collaboration, and accountability. We all have to push the envelope and practice at the top of our scopes in order to make health care better every day. One of the ways we might be able to do that now is by considering these new high-sensitivity troponins as a useful new clinical tool to improve patient outcomes.

Thanks for reading! See you next time!

Disclosure: I am no longer affiliated with Swedish Covenant Hospital in Chicago as an employee, and any recount of policy and/or procedure(s) specifically regarding their cardiology protocols and troponin resulting are a historical and anecdotal account of my time working there in the past. I have no affiliations with SIEMENS, Advia, Roche, or any other medical laboratory instrumentation institution. I am only affiliated with Bronx Care Hospital System as a current rotating medical student and my account of their transition to 5th gen testing is anecdotal from discussions with in-house staff, cardiologists, and laboratory management.

 

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Constantine E. Kanakis MSc, MLS (ASCP)CM graduated from Loyola University Chicago with a BS in Molecular Biology and Bioethics and then Rush University with an MS in Medical Laboratory Science. He is currently a medical student at the American University of the Caribbean and actively involved with local public health.

Tissue is the Issue: Direct Observation of Microorganisms (part 2)

Most laboratories (and clinicians) utilize and rely on microscopic observation as the first step in the detection and identification of microorganisms. In some cases, direct microscopic analysis is used to determine the immediate clinical course of action. For example, if during a surgical procedure infection is suspected, then it is possible for the surgeon to submit a specimen to the laboratory for rapid (STAT) analysis. If polymorphonuclear leukocytes (PMN) and organisms are observed, then the differential diagnosis of infection is confirmed. Assuming proper specimen collection; if no PMN and no organisms are seen, then infection is less likely (true in most cases; there are always exceptions). These direct microscopic observation results thus drive the surgeon’s decision to either remove the infected area or perhaps advises on the use of intraoperative antimicrobials.

In part 1 of this series we discussed the pre-analytical problems associated with tissue culture; specifically, how specimen processing can affect whether or not organism is detected and recovered (1). In part 2 we will consider methods used for the direct observation of microorganisms in tissue specimens. 

The Issue

Let’s take a step back to review our previous “culture-negative” endocarditis case (1). Recall that the blood cultures were negative at first, no organisms were observed on the Gram stain and the culture was also negative of the valve tissue. However, when we evaluated the frozen tissue that was “split” and saved for sequencing, the organism was observed via acridine orange (AO).

AO.png
Image 1. AO of a positive broth culture from a homogenized tissue specimen. The AO stain displays cocci, bacilli, and yeast. The DNA-containing cells fluoresce orange.

 The Solution

AO is a fluorescent dye that intercalates nucleic acids. It is a rapid, inexpensive, and most importantly- it is a sensitive alternate dye that can aid in microorganism detection in a variety of specimen types (2, 3 ,4, 5). AO is more specific than Gram stain (2, 3). Because bacterial DNA is not contained in a nucleus, but freely contained within the cell, the AO-stained cell takes the shape of the organism’s cellular morphology (Image 1). For example, if the organism is Staphylococcus aureus, then the AO would exhibit Gram positive cocci in clusters. Similarly, if the organism in question is Escherichia coli, then Gram negative bacilli would be observed, etc.

Some organisms (Campylobacter, Mycoplasma, etc.) do not stain well with Gram stain and as such they can be difficult to detect in direct specimen observations. The presence of many PMN and no organisms may be a clue that the organism is present in low numbers or that the organism does not stain well with the Gram stain. In cases such as these, the use of AO has proven to be very useful. Additionally, if the morphology of an organism in a Gram stain is difficult to interpret, then AO can also provide a more clear-cut answer. Lastly, the presence of artifact(s) can be problematic when reading Gram stains. “Is that Gram positive cocci or junk?”. Junk usually does not contain DNA and therefore does not fluoresce. Therefore, the information provided by the AO stain can aid in your decision to report cocci or not.

One disadvantage is that a fluorescent microscope is required to visualize the stain. Implementing AO may require the purchase of new equipment as not all laboratories have access to a fluorescent microscope.  Because AO stains nucleic acid, anything with DNA or RNA will stain positive. This is another disadvantage because too much material that is positively stained can make interpreting the stain difficult at times. Another disadvantage is that a Gram stain is still required because the AO only allows the visualization of DNA-containing cells, it does not determine the Gram stain reaction.

Thinking back to our original case, the organism was present in the frozen tissue piece. It was not detected in the direct Gram stain, but rare organisms were noted in the AO. Because the AO was positive, we then reviewed the Gram stain and found rare Gram negative bacilli. This suggests that the organism was initially missed in the Gram stain because of the low abundance present. Bottom line, the AO was positive and we were thus able to provide the clinical team with relevant information.

The Conclusion

There are many stains used to aid in the observation of microorganisms. The AO stain is easy to perform, inexpensive, rapid, sensitive, and versatile. AO can be used on direct specimen smears, isolated colonies, and formalin-fixed paraffin embedded sections. Although there are limitations to using AO, the benefits considerably outweigh the shortcomings. AO is a great tool that laboratories should consider implementing as an alternate method for the direct observation of microorganisms.

References

  1. https://labmedicineblog.com/2018/04/20/tissue-is-the-issue-splitting-specimens-part-1/
  2. Mirrett, S., Lauer, B.A., Miller, G.A., and Reller, B. 1982. Comparison of Acridine Orange, Methylene Blue, and Gram stains for Blood Cultures. J. Clin Microbiol. 15; 4: 562-566.
  3. Lauer, B.A., Reller, B., and Mirrett, S. 1981. Comparison of Acridine Orange and Gram Stains for Detection of Microorganisms in Cerebrospinal Fluid and Other Clinical Specimens. J. Clin Microbiol. 14; 2:201-205.
  4. Martinez, R.M., Bowen, T.R., and Foltzer, M.A. Prosthetic Devise Infections. Diagnostic Microbiology of the Immunocompromised Host. 2016 (Book chapter, chapter 27. Pages 711- 733. ASM Press.
  5. Cooper, J.D., Dometita, D., Hasan, A., Dorion, P., Wolk, D.M. and Martinez, R.M. Orange you glad you checked the buffy coat? Clin. Microbiol. Newslett. 37: 9-13.

 

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-Raquel Martinez, PhD, D(ABMM), was named an ASCP 40 Under Forty TOP FIVE honoree for 2017. She is one of two System Directors of Clinical and Molecular Microbiology at Geisinger Health System in Danville, Pennsylvania. Her research interests focus on infectious disease diagnostics, specifically rapid molecular technologies for the detection of bloodstream and respiratory virus infections, and antimicrobial resistance, with the overall goal to improve patient outcomes.

Hematopathology Case Study: A 16 Year Old Male with Fatigue, Fevers, and Weight Loss

Case History

16 year old male with a history of chronic pilonidal cyst presented with fatigue, fevers and weight loss. He was febrile and noted to have cervical and inguinal adenopathy. Labs were significant for a white count of 77,000 with 85% peripheral blasts, anemia and thrombocytopenia.

MPAL1
Bone marrow aspirate
MPAL2
Bone marrow core biopsy
MPAL3.png
Flow cytometry myeloid markers
MPAL4
Flow cytometry cytoplasmic markers
MPAL5
Flow cytometry T-cell markers

Diagnosis

The bone marrow aspirate shows cellular spicules with sheets of intermediate-to-large sized mononuclear cells with irregular nuclei, distinct nucleoli, dispersed chromatin, and scant to generous amphophilic cytoplasm, with occasional vacuoles, consistent with blasts.

The bone marrow core biopsy shows a greater than 95% cellular marrow, hypercellular for age with approximately 90% of the cellularity composed of an interstitial population of intermediate-to-large sized mononuclear cells with irregular nuclei, distinct nucleoli, dispersed chromatin, and scant to generous amphophilic cytoplasm, with occasional vacuoles, consistent with blasts.

Flow cytometry shows leukemic cells that express immaturity markers (TdT, CD34, CD117, HLA-DR), T cell lineage markers (CD2, CD7 cCD3), and multiple myeloid markers (CD13, CD117, and variable CD15 and CD11b as well as MPO in a small subset).

Bone marrow core biopsy staining (not shown) had similar findings with blasts showing dim-to-strong positivity for myeloperoxidase, lysozyme, CD34 and CD117, as well as strong positivity for TdT. CD7 was weakly positivity, as well as CD3. CD4 and CD5 were negative.

MPAL6
Genetics diagnostics
MPAL7
NGS panel

With the expression of MPO by flow cytometric analysis and immunohistochemistry, a final diagnosis of acute leukemia with myeloid and T lymphoid phenotypic features, most consistent with T/Myeloid Mixed Phenotype Acute Leukemia (MPAL) was rendered. 

Discussion

Most acute leukemias are definitively assigned to either myeloid, T or B lymphoid lineages. However, approximately 2-5% of patients diagnosed with acute leukemia display an ambiguous lineage after immunophenotyping. A portion of these cases are classified under the category of mixed phenotype acute leukemia (MPAL) by the current WHO nomenclature.1

In a study of 117 MPAL patients by Yan et al, 55% of the cases had combined B/Myeloid, while 33% had T/Myeloid, and 12% had B/T/Myeloid. CD34 was strongly positive in 82% of cases, which reinforces the idea that the cell of origin is a multi-potent stem cell capable of differentiating into both myeloid and lymphoid progenitors. Cytogenetic analysis revealed no chromosomal abnormality in 36% of the patients with MPAL, while 64% had complex karyotypes (>3 aberrations). Translocation (9;22) was the most common abnormality, found in 15% of patients. Monosomy 7, a common finding in myelodysplastic syndromes as well, was found in 7.6% of patients. Mutational analysis revealed IKZF1 deletions in 13% of patients, ASXL1 in 6.5% of patients and a variety of other mutations including ETV6, NOTCH1 and TET2.2

In 2016, Eckstein and colleagues demonstrated epigenetic regulatory genes such as DNMT3A, IDH2, TET3 and EZH2 are the most commonly mutated in MPAL. RAS mutations including NRAS and KRAS and tumor suppressors, such as TP53 and WT1, were frequently identified as well.3

Interestingly enough, the genetic features of MPAL often overlap with early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). ETP-ALL is a high-risk subgroup, representing 10% of adult T-lineage acute lymphoblastic leukemia. It is defined by a characteristic immunophenotype (CD1a/CD8 negative with weak CD5) and distinct gene expression associated with early arrest in T-cell development. This subgroup, called the LYL1 group, expresses the early hematopoietic marker CD34 as well as myeloid antigens (CD13 or CD33), but lacks expression of both CD4 and CD8. These leukemias are associated with a poor prognosis, with a 10- year overall survival of 19% compared to 84% for all other T-ALLs.4

Zhang et al in 2012 performed whole genome sequencing on ETP-ALL cases and found a high frequency of mutations in factors mediating cytokine receptor, tyrosine kinase and RAS signaling. It also showed inactivating mutations in genes encoding transcription factors (GATA3, ETV6, RUNX1, IKZF1) as well as genes involved in histone modification, such as EZH2.5

Overall, the genetic features of both ETP-ALL and MPAL display an identical genomic pattern that involves multiple pathways, including tyrosine kinase signaling, cytokine receptor response, RAS pathway activation, and loss of function in tumor suppressors. These findings give credence to the hypothesis that the early T-cell precursor actually displays more of a pluripotent stem cell profile that is similar to myeloid neoplasms, thus confounding findings found during molecular profiling. With this paradigm in mind, molecular diagnostics cannot differentiate between ETP-ALL and in this case, MPAL.

 

References

  1. Swerdlow, Steven H. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4th ed., International Agency for Research on Cancer, 2017.
  2. Yan et al. Clinical, immunophenotypic, cytogenetic, and molecular genetic features in 117 patients with mixed-phenotype acute leukemia defined by WHO-2008 classification. 2012 November;97(11):1708-12.
  3. Eckstein OS et al. Mixed Phenotype Acute Leukemia (MPAL) Exhibits Frequent Mutations in DNMT3A and Activated Signaling Genes. Exp Hematol. 2016 August; 44(8):740-744.
  4. Ferrando AA et al.  Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia. Cancer Cell. 2002. 1:75–87.
  5. Zhang J et al. The genetic basis of early T-cell precursor acute lymphoblastic leukemia. Nature. 2012 Jan 11;481(7380):157-63.

 

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Chelsea Marcus, MD is a third year resident in anatomic and clinical pathology at Beth Israel Deaconess Medical Center in Boston, MA and will be starting her fellowship in Hematopathology at BIDMC in July. She has a particular interest in High-grade B-Cell lymphomas and the genetic alterations of these lymphomas.