Hematopathology Case Study: What’s in Those Histiocytes?

Case history

A 50 year old female with a past medical history significant for Sjogren’s syndrome and ventricular tachycardia s/p ICD placement presented for a routine chest X-ray in which a 1.8 cm spiculated left upper lobe lung mass was identified. A subsequent PET scan revealed FDG avidity. Other Imaging revealed no lymphadenopathy. The patient is a non-smoker and has no other comorbidities. A core needle biopsy with fiducial placement was performed.

Diagnosis

histio1
H&E 10x
histio2
H&E, 20x
histio3
H&E, 50x
histio4
CD3
histio5
CD20
histio6
CD79a
histio7
IgG
histio8
CD68
histio9
CD138
histio10
Kappa ISH
histio11
Lambda ISH

Sections of lung core biopsy material show numerous histiocytes containing eosinophilic intracytoplasmic globular inclusions. An admixed population of plasma cells are seen which are present in aggregates along with mature appearing lymphocytes. The plasma cells also demonstrate globular inclusions within their cytoplasm.

By immunohistochemistry, CD3 highlights scattered mature T-cells while CD20 highlights B-cells present in focal aggregates. Numerous plasma cells are present and are positive for CD138, CD79a, BCL2, and MUM1. By in situ hybridization, plasma cells are greatly kappa predominant. IgG is positive in the majority of the plasma cells with only rare cells staining for IgA and IgM. CD68 is positive in the numerous histiocytes.

IGH gene rearrangement studies by PCR demonstrated was positive, indicating a clonal population.

Overall, the findings are consistent with a crystal-storing histiocytosis with an associated plasma cell neoplasm or low-grade B-cell lymphoproliferative disorder.

Following the diagnosis, the patient received stereotactic body radiation therapy given the localized findings.

Discussion

In this case, the findings are morphologically consistent with crystal-storing histiocytosis (CSH), which is a rare lesion that is the result of intralysosomal accumulation of immunoglobulin. The immunoglobulin is stored as crystalline structures within histiocytes that occupy the vast majority of a mass forming lesion. Multiple sites can be involved, which include bone marrow, lymph nodes, liver, spleen, gastrointestinal tract, and kidney. Most often, the lesion is confined to a single site but occasional generalized forms with multiple organ involvement have been described. CSH is also often associated with B-cell lymphoproliferative disorders or plasma cell dyscrasias, but rarely are the result of chronic inflammatory conditions.

The assessment of CSH requires excellent staining to identify the quality of the histiocytes. As mentioned, CSH will show intracytoplasmic inclusions that are eosinophilic in nature. Mimickers of CSH include mycobacterial and fungal infections, mycobacterial spindle cell pseudotumor, malakoplakia, HLH, storage disorders such as Gaucher’s, as well as histiocytic lesions such as xanthogranuloma, Langerhans cell histiocytiosis, fibrous histiocytoma, Rosai Dorfman disease and rarely other eosinophilic tumors such as rhabdomyoma, granular cell tumor, and oncocytic neoplasms.1

A thorough review of the literature as well as a clinicopathologic study by Kanagal-Shamanna R et al revealed that the localized type of CSH was the dominant presentation in which over 90% of cases showed isolated masses. Per previous reviews, localized lesions were often found in the head and neck as well as lung.2 A study group in which 13 cases that showed CSH, 12 demonstrated an underlying lymphoma or plasmacytic neoplasm. Interestingly, in 5 of the cases, the histiocytic infiltrate was so prominent and dense that it obscured the underlying neoplasm. In these particular cases, immunohistochemistry and PCR were of great importance.

Although the majority of cases of CSH are the result of an underlying lymphoproliferative disorder or plasma cell neoplasm, rare cases of report inflammatory processes have been described, particularly in the setting of an immune mediated process such as rheumatoid arthritis or Crohn disease.

Overall, although a rare entity, it is important to be aware of CSH and its mimickers as this can be an elusive diagnosis to make, especially when the histiocytic infiltrate is dense.

References

  1. Kanagal-Shamanna R, et al. “Crystal-Storing Histiocytosis: A Clinicopathologic Study of 13 Cases,” Histopathology. 2016 March; 68(4): 482-491.
  2. Dogan S, Barnes L, Cruz-Vetrano WP “Crystal-storing histiocytosis: a report of a case, review of the literature (80 cases) and a proposed classification,” Head Neck Pathol. 2012; 6:11-120.

 

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-Phillip Michaels, MD is a board certified anatomic and clinical pathologist who is a current hematopathology fellow at Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. His research interests include molecular profiling of diffuse large B-cell lymphoma as well as pathology resident education, especially in hematopathology and molecular genetic pathology.

Groupthink and the Abilene Paradox

Everyone makes decisions and everyone makes decisions in groups. But we all have been part of groups that felt anything but productive: the end result was subpar, the process took a lot longer than if you had done it yourself, and the endless arguments and indecision caused you and everyone else on the team a lot of stress.

So how can we ensure that an effective group decision-making process is in place? How can we be certain that the decision we make as a group are the right ones?

Groupthink is when group members’ desire for harmony and unanimity overrides their motivation to realistically consider alternative courses of action. Not considering alternatives can have severe consequences as this additional information could have altered an idea or process to make it more effective. An example of Groupthink is with the company Theranos, where the entire board went along with Holmes’ ideas without looking for alternative solutions. Needless to say, they would have benefitted from those alternative options. Groupthink occurs because members of a group do not feel comfortable sharing their dissenting opinions due to high group cohesiveness, stressful situations, and a feeling that the group will always be successful.

The Abilene Paradox complements the Groupthink concept: with Groupthink it is a few members of a group that do not speak up, while with the Abilene Paradox the majority (or the entire group) remains silent. Furthermore, with Groupthink even the dissenting (and silent) member will usually feel good about the final decision, while with the Abilene Paradox members feel resentment. The reasons the Paradox occurs revolve around fears of separation and reprimand. The results are group projects that never go anywhere or fail, and separation from the group.

The good news is that both situations can be avoided by one main thing: creating an open environment in which dissenting opinions are encouraged, listened to, and incorporated. So next time you are in a group or meeting, pay attention to the number of dissenting opinions stated and how people respond to them. Reflect on how you respond to those opinions and if there is something you can do to increase the openness in your team or department.

lotte-small

-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.


 

In pursuit of efficiency and continuous improvement, the pathologists, residents and technical staff at Makerere University, meet every Wednesday. They are also joined by colleagues from the Mulago National Referral Hospital under the Ministry of Health of Uganda.  The purpose of this meeting is to make decisions that pertain to teaching, operation of the histopathology service, and conducting research.

Since 2003, I have attended several of these meetings. However, the realization and achievement of goals has been poor and nearly zero in some instances. No prior agenda is sent out. During the meetings, counter-productive decisions are made. The generation of ideas, alternatives and creativity has dwindled meeting after meeting. Attendance and contribution to meetings are viewed as a waste of time and effort. This has led to low motivation, and attendance has hit an all-time low.

There is always somebody to blame for the failure in teaching, the inefficient biopsy service and the low research productivity at the end of each year. In the last team building workshop, the Mulago team did not show up despite an earlier commitment and receiving an invitation three weeks to time. In the first workshop, the executive director of Mulago did not show up even when he was scheduled to speak! We have had a share of failed projects and ventures; the FNA clinic and teaching of Cytotechnology have not gone well, establishment of the “pay for service” laboratory is struggling and embedding QA and QC systems in the histopathology laboratory has been a total failure. Lately, we lost the bid to the ASCP Cancer Diagnostics for the Africa telepathology project.

In summary, the department seems to be stuck in thick mud! What is in play here?  It is the groupthink and Abilene paradox; the making of irrational decisions and failure to manage group agreement respectively.  This is an expected natural occurrence in decision making involving groups.

The motivation for being on the ASCP leadership academy is my passion for change through leadership. That is what drives me. I love effective participation and being part of productive teams.

Following the completion of the two courses, I now clearly understand what has been happening in my department. I look back and easily recognize the symptoms of groupthink; the illusion of unanimity, self-censorship, mind guards, and shared stereotypes.

Recognition of the problem is half way to the solution. Techniques to avoid the problem are now known to me. The style of meetings in the department needs to be changed. The chair should avoid being too directive in order to ensure an open climate during discussions.  The chair should also assign a member the role of a critical evaluator and once in a while open up the meetings to important outsiders.

All the decisions to do the above had to be agreed upon by members during the weekly meetings. I now recognize and understand the play of the Abilene paradox here; that some members said “yes” when indeed they meant to say “no.” I look back and recognize action anxiety, fear of separation, real risk, negative fantasy, perceived risk and confusion of risk and certainty.

Looking at the 15 reasons as to why one ends on the road to Abilene, I can surely identify with each of them. This, I am sure, is shared by each of the staff members.

We need to avoid the trip to Abilene by making changes in the conduct of meetings. We should plan before the meetings, invite the right people, clearly state the decisions to be made, plan enough time for discussion, and set a climate of openness. During the meetings, vital questions should be asked. It takes a lot to plan but in the end, the returns are long term and a good on time invested.

What can be done differently now? Sharing the new knowledge learned during the course is the first step. The second step is to put into practice the techniques to avoid the group think and endless trips to Abilene. These must eventually become the new culture of practice and eventually spread across other university departments.

RL

-Robert Lukande is trained as an anatomical pathologist and is currently employed at the College of Health Sciences, Makerere University, Kampala Uganda. He is a teacher, research and consultant, and is passionate about changing the lives of others through leadership. He’s the current president of APESCA and is acting chair of the Department of Pathology.

Microbiology Case Study: A 7 Month Old Female with Fever and Seizure-Like Episodes

Case History

A 7 month old female presented to the emergency department (ED) due to fever and seizure-like episodes. Her mother reported the child had been persistently febrile for 5 days (Tmax 103.9°F) with rhinorrhea, fussiness and decreased oral intake. The patient experienced 3 seizure-like episodes on the day of admission, which the mother described as periods of “shaking” with eyes rolling back. The child was unresponsive during these episodes, which lasted 1 to 2 minutes each. The child had been taken to her pediatrician the day prior to presentation to the ED where she was given a shot of ceftriaxone for presumed otitis media. The child received a chest x-ray, influenza testing, and blood and urine cultures were collected. She also had a lumbar puncture performed and the cerebral spinal fluid (CSF) was sent for chemistries, bacterial culture and polymerase chain reaction (PCR) testing for meningitis/encephalitis pathogens. She was started on IV ceftriaxone.

Laboratory Testing

The child’s white blood cell count from peripheral blood was 7.1 TH/cm2 and chest x-ray, urinalysis and flu testing were unremarkable. The CSF was clear and colorless with 7 WBC/cm2, glucose of 57 mg/dL and protein of 21 mg/dL. The cytospin Gram stain identified no organisms. The meningitis/encephalitis panel detected the presence of human herpesvirus 6 (HHV-6).

Discussion

Human herpesvirus 6 is a member of the Herpesviridae family and was the sixth herpes virus identified. Structurally, HHV-6 possesses a double stranded DNA genome and is enveloped. Clinically, it is the etiologic agent of roseola infantum (exanthum subitum) in infants and toddlers. Primary infection occurs in early childhood and those infected can be asymptomatic or have a non-specific febrile illness while only the minority present with the characteristic red macular rash prominent on the trunk and extremities, lymphadenopathy and high fevers. HHV-6 is highly neurotropic and as such causes viral encephalitis with 5-15% of children experiencing febrile seizures as a result of this illness. HHV-6 is highly prevalent with a greater than ninety percent seropervalence rate. HHV-6 establishes latency in T lymphocytes and can reactivate & cause disease, especially in immunocompromised patients such as those recipients of stem cell or solid organ transplants.

Traditional laboratory methods of identification for HHV-6 were challenging as viral culture, while once the gold standard for active disease, is not practical for most labs and is no longer used in routine diagnostics. PCR from serum, plasma or CSF has become the preferred test as there are now FDA-cleared, commercial platforms that are easy to use, allow for rapid turnaround time and in the case of multiplex PCR panels, the ability to target multiple pathogens from one test. Serology, while helpful in the diagnosis of primary infections, may not be provide conclusive results in a timely manner and is of limited utility in reactivation. Other less commonly used methods include immunohistochemistry, in situ hybridization and electron microscopy.

The prognosis for patients infected with HHV-6 is generally good with self-limited illness not requiring treatment. Rarely, multi-organ involvement can occur and HHV-6 infection in immunosuppressed patients can be a major cause of morbidity and mortality. There is no antiviral therapy licensed for the treatment of reactivated disease in this setting, but approaches using ganciclovir and valganciclovir have been proposed.

In the case of our patient, her blood, urine and CSF cultures were negative and her antibiotics were stopped after cultures were no growth at 24 hours. She required no treatment other than supportive care with acetaminophen for fever control. Prior to discharge, she developed a fine rash on her face, the back of her neck and trunk that was characteristic of an HHV-6 rash. This case demonstrates the utility of multiplex PCR testing in providing rapid identification of pathogenic organisms allowing for real time diagnosis and the limiting of unnecessary treatment.

 

ET

-Eric Tillotson, MD, is a second year Anatomic and Clinical Pathology resident at the University of Mississippi Medical Center.

Stempak

-Lisa Stempak, MD, is an Assistant Professor of Pathology at the University of Mississippi Medical Center in Jackson, MS. She is certified by the American Board of Pathology in Anatomic and Clinical Pathology as well as Medical Microbiology. She is the director of the Microbiology and Serology Laboratories. Her interests include infectious disease histology, process and quality improvement and resident education.

 

 

Does Price Transparency Improve Lab Utilization?

Physicians often have poor awareness of costs. For that reason, many believe that providing cost information to physicians would increase awareness that, in turn, could improve laboratory utilization. For example, costs of lab tests could be displayed as a field in the computerized provider order entry system. Interventions of this type are attractive because they are relatively inexpensive to implement and do not disrupt workflow with popups. Further, unlike other interventions, cost display is sustainable. Some interventions require constant training and followup whereas cost display is a one-time intervention. For these reasons, organizations are experimenting to see the effect of cost display on laboratory utilization.

Does cost display reduce lab utilization? Studies have shown wide variation in impact. Most studies have focused on orders for laboratory testing and imaging; however, a few studies have looked a pharmaceuticals.  A recent systematic review concluded that cost display is associated with a modest reduction in laboratory utilization.(1) The review included twelve studies on lab utilization and all of these showed improvement.(2-13) However, a more recent study by Sedrak et al. found that cost-display had no impact on utilization.(14) Similarly, two imaging studies found that cost-display had no effect on orders.(4, 15). There was a wide variation in impact: test utilization reduction ranged from 0% to over 30% in some cases. Overall, it appears that cost display tends to reduce utilization; however, it sometimes has no effect as shown in the Sedrak study. So far, cost display has never been associated with an increase in utilization. We have experimented with cost display at University of Utah and, like the Sedrak study, found no effect.

Why is there such a range of effects? Can we predict which organizations are likely to benefit? The short answer is that nobody knows.  The twelve studies on lab utilization where conducted in a wide range of settings (community, academic and pediatric hospitals), included different numbers of tests, or had other differences that could affect results. The way in which costs are displayed also varies. Some sites use the Medicare Maximum Allowable Reimbursement Rate, some use a series of dollar signs to indicate cost categories, and others use charges. It is not clear whether these differences matter.

There are a number of factors that might affect the impact of cost display. For example, cost display might have less impact at an institution that has an effective utilization management program in place because there is less opportunity for improvement. Or, the number of tests with costs displayed may have an impact. For example, some studies have displayed costs for a relatively few number of tests whereas other studies showed costs for a large number of tests.  Cost display for a few tests may send a different signal to providers than providing costs for all tests. Also, we don’t know how long the intervention works. Is there an initial effect that wears off? If so, how long does it last? These questions will need to be resolved by future studies.

In the meantime, should you provide cost feedback at your institution? It is hard to predict what will happen but most evidence suggests that you will see some improvement in utilization. It is not expensive to implement and some organizations have seen a significant impact. At worst, the evidence suggests that you will see no effect on testing behavior.  On balance, cost-display seems like a low-risk intervention.

 

References

  1. Silvestri MT, Bongiovanni TR, Glover JG, Gross CP. Impact of price display on provider ordering: A systematic review. Journal of Hospital Medicine 2016;11:65-76.
  1. Fang DZ, Sran G, Gessner D, et al. Cost and turn-around time display decreases inpatient ordering of reference laboratory tests: A time series. BMJ Quality and Safety 2014;23:994-1000.
  1. Nougon G, Muschart X, Gérard V, et al. Does offering pricing information to resident physicians in the emergency department potentially reduce laboratory and radiology costs? European Journal of Emergency Medicine 2015;22:247-52.
  1. Durand DJ, Feldman LS, Lewin JS, Brotman DJ. Provider cost transparency alone has no impact on inpatient imaging utilization. Journal of the American College of Radiology 2013;10:108-13.
  1. Feldman LS, Shihab HM, Thiemann D, et al. Impact of providing fee data on laboratory test ordering: A controlled clinical trial. JAMA Internal Medicine 2013;173:903-8.
  1. Horn DM, Koplan KE, Senese MD, Orav EJ, Sequist TD. The impact of cost displays on primary care physician laboratory test ordering. J Gen Intern Med 2014;29:708-14.
  1. Ellemdin S, Rheeder P, Soma P. Providing clinicians with information on laboratory test costs leads to reduction in hospital expenditure. South African Medical Journal 2011;101:746-8.
  1. Schilling UM. Cutting costs: The impact of price lists on the cost development at the emergency department. European Journal of Emergency Medicine 2010;17:337-9.
  1. Seguin P, Bleichner J, Grolier J, Guillou Y, Mallédant Y. Effects of price information on test ordering in an intensive care unit. Intensive Care Medicine 2002;28:332-5.
  1. Hampers LC, Cha S, Gutglass DJ, Krug SE, Binns HJ. The effect of price information on test-ordering behavior and patient outcomes in a pediatric emergency department. Pediatrics 1999;103:877-82.
  1. Bates DW, Kuperman GJ, Jha A, et al. Does the computerized display of charges affect inpatient ancillary test utilization? Arch Intern Med 1997;157:2501-8.
  1. Tierney WM, Miller ME, McDonald CJ. The effect on test ordering of informing physicians of the charges for outpatient diagnostic tests. N Engl J Med 1990;322:1499-504.
  1. Everett GD, Deblois CS, Chang PF. Effect of Cost Education, Cost Audits, and Faculty Chart Review on the Use of Laboratory Services. Arch Intern Med 1983;143:942-4.
  1. Sedrak MS, Myers JS, Small DS, et al. Effect of a Price Transparency Intervention in the Electronic Health Record on Clinician Ordering of Inpatient Laboratory Tests: The PRICE Randomized Clinical Trial. JAMA Internal Medicine 2017.
  1. Chien AT, Ganeshan S, Schuster MA, et al. The effect of price information on the ordering of images and procedures. Pediatrics 2017;139.

 

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-Robert Schmidt, MD, PhD, MBA, MS is a clinical pathologist who specializes in the economic evaluation of medical tests. He is currently an Associate Professor at the University of Utah where he is Medical Director of the clinical laboratory at the Huntsman Cancer Institute and Director of the Center for Effective Medical Testing at ARUP Laboratories.

Microbiology Case Study: A 56 Year Old Woman with Purulent Vaginal Discharge and Dysarthria

Case History

The lab received a purulent fluid from a frontal subdural empyema in a 56 year old woman with a several month history of sporadic bloody to purulent vaginal discharge and a two week history of severe headache, facial weakness, and recent dysarthria causing her to seek treatment. Pelvic exam and CT of the abdomen revealed a fungating cervical mass, later found to be adenocarcinoma, with possible fluid collection within the uterine cavity.

Lab Identification

Initial gram stain of the subdural fluid showed moderate neutrophils and no bacteria. The aerobic plate showed no growth at 48 hours. The thioglycollate broth grew “puffballs” containing long gram negative bacilli with tapered ends.

 

fusnuc1
Anaerobic blood agar plate on day 9. Note: large white colonies are contamination.
fusnuc2
Gram stain from anaerobic plate on day 9.

The anaerobic plate grew few grey colonies composed of similarly-shaped gram negative bacilli. MALDI-TOF mass spectrometer identified both colony morphologies as Fusobacterium nucleatum.

Discussion

F. nucleatum is a gram negative, spore non-forming, asacchrolytic, slow-growing, obligate anaerobe that can fluoresce chartreuse under UV light. It is one of 14 species within the Fusobacterium genus and is further classified into 5 subspecies (nucleatum, animalis, fusiforme, vincentii, and polymorphum) which have different pathogenic profiles. It is commonly associated with the oral cavity, though it is unclear if it is ever a constituent of usual flora. Its main virulence factors are invasion of epithelial and endothelial cells, induction of host immune response, and adhesion to tissue through a variety of adhesins. The FadA adhesin interacts with the ubiquitous cell junctional cadherins leading to increased permeability of the epithelial and endothelial barrier. This interaction may be why F. nucleatum infections can be found in such diverse locations within the body and are often part of a polymicrobial infection [1, 2].

F. nucleatum is a constituent of oral plaque and is strongly associated with gingivitis and periodontitis. It is present in increased quantity with increasing severity of disease and in patients with a history of smoking or uncontrolled diabetes mellitus. It is a known pathogen in infections and abscesses of the head and neck, brain, lung, abdomen, blood, and pleura. It is also commonly found in placental and fetal tissues and strongly associated with a variety of obstetrical conditions including preterm birth, chorioamnionitis, and neonatal sepsis. It has been implicated in at least one case as the causative agent of stillbirth. Its prevalence in cord blood in cases of neonatal sepsis is equal to or greater than that of E. coli and Group B Strep. There is also a known association between F. nucleatum and colorectal cancer and IBD, with current research investigating whether the bacteria could play a role in pathogenesis. [1].

F. nucleatum is generally responsive to treatment with a range of antimicrobials, though there are reports of strains resistant to clindamycin and beta-lactamase-based resistance to ampicillin [2, 3].

The infectious disease clinician covering the present case suggested that the patient may have been transiently bacteremic due to her fungating gynecologic malignancy and suffered a minor head trauma causing a small subdural hemorrhage that seeded the bacteria in a sufficiently protected anaerobic environment.

 

References

  1. Han TW. Fusobacterium nucleatum: a commensal-turned pathogen. Curr Opin Microbiol. (2015) 23:141-147.
  2. Denes E, Barraud O. Fusobacterium nucleatum infections: clinical spectrum and bacteriological features of 78 cases. Infection (2016) 44:475-481.
  3. Veloo ACM, Seme K, Raanges E, Rurenga P, Singadji Z, Wekema-Mulder G, van Winkelhoff AJ. Antibiotic susceptibility profiles of oral pathogens. Intl J Antimicrob Agents. (2012) 40:450-454.

 

-Taylor Goller is a Pathology Student Fellow at University of Vermont Medical Center.

Wojewoda-small

-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 16 Year Old with Rhinosinusitis

Case

A 16-year-old male presented with recurrent sinusitis and rhinitis. He had a history of left sinus surgery two years ago, and at that time pathologic examination of the tissue demonstrated eosinophilia and fungal culture grew Curvularia, consistent allergic fungal sinusitis. The patient was doing well without allergy or immunotherapy management until three months ago when he could not breathe out of the right nostril and began snoring loudly. He underwent bilateral endoscopic frontal sinusotomy with tissue removal of the ethmoid and sphenoid sinuses. Tissue was sent to the laboratory for fungal culture. After five days, fungal cultures grew mold on inhibitory mold agar with gentamicin. The surface was a gray speckled color (Image 1C). The reverse color of the mold colony was dark brown to black. The microscopic appearance can be seen in Image 1A-B.

Bipolaris figure
Image 1. A) Ellipsoid conidia with the common number of 3-5 septations stained with lactophenol cotton blue counterstain, B) a cluster of conidia surrounded by less lactophenol cotton blue stain better demonstrating brown melanin pigment in the cell wall, and C) a dark gray speckled fungal colony.

 

Discussion

These features are consistent with the identification of Bipolaris. Microscopic examination using lactophenol cotton blue tape prep demonstrated oblong conidia characteristic of Bipolaris (Image 1 A-B). The conidia are ellipsoidal with pale brown pseudoseptations that contain three to five septa. Four septa are the most common. Bipolaris is a dematiaceous fungus, meaning the cell walls contain dark brown melanin pigment. This can be seen by microscopic observation of the fungal cell wall which contains pigment (Image 1B) and is also demonstrated by the dark reverse color of the fungal colony.

To distinguish Bipolaris from Drechslera and Exserohilum, the Germ tube test can be utilized. When conidia are incubated with a drop of water on a glass slide for 8-24 hours, they will begin to form Germ tubes. Bipolaris species germinate from both poles of the oblong conidium at a 180 degree angles (hence the name “Bipolaris”), whereas Exserohilum germinate from just one pole at a 180 degree angle and Dreschslera species germinate at a 90 degree angle from the central cells of the conidium. Dreschslera can be confused for Bipolaris based on colony appearance and microscopic appearance, but unlike Bipolaris, Dreschslera is not associated with human disease.1

Pathogenic strains of Bipolaris include Bipolaris australiensis, Bipolaris hawaiiensis, Bipolaris maydis, Bipolaris melanidis, Bipolaris speicifera, and Bipolaris sorokiniana.2 The most common cause of infection is Bipolaris spifcifera. Bipolaris species are the most common cause of fungal sinusitis in immunocompetent individuals which often presents as allergic rhinitis. Allergic rhinitis could also be a risk factor for acquiring Bipolaris. Treatment often consists of prompt surgical excision to prevent expansion, superficial deformity and dissemination. If fungal chemotherapy is pursued, itraconazole and amphoterin B have been reported as effective agents.3

Bipolaris is one of the most common causes of allergic fungal sinusitis, typified by nasal polyps and mucus plugs consisting of eosinophils, fungal hyphae and Charcot-Leyden crystals. It is a type 1 and 3 hypersensitivity reaction mediated process due to high levels of mold-specific IgE.4 Skin prick testing is also positive in patients with allergic fungal rhinosinusitis (AFRS) which further indicates that the pathophysiology is an immunologic versus infectious process.4 While the exact process of fungal allergic sensitization has not been codified, chitin, a structural fungal protein has been shown to elicit a Th2 immune response.5 It will be interesting to see how this research evolves so that we might one day see why fungi can cause both erosive infections and allergies within human patients.

 

References

  1. Fothergill AW. Identification of Dematiaceous Fungi and Their Role in Human Disease. Clin Infect Dis. 1996; 22 (S2): S179-84.
  2. Shafili SM, Donate G, Mannari RJ, Payne WG, Robson MC. Diagnostic Dilemmas: Cutaneous Fungal Bipolaris Infection. Wounds. 2006; 18(1):19-24.
  3. Saenz RE, Brown WD, Sanders CV. Allergic Bronchopulmonary Disease Caused by Bipolaris hawaiiensisPresenting as a Necrotizing Pneumonia: Case Report and Review of Literature. The American Journal of Medical Sciences. 2001; 321(3):209-12.
  4. Manning SC, Holman M. Further evidence for allergic pathophysiology in allergic fungal sinusitis. Laryngoscope. 1998;108(10):1485–1496.
  5. Reese TA, Liang HETager AMLuster ADVan Rooijen NVoehringer DLocksley RM. Chitin induces accumulation in tissue of innate immune cells associated with allergy. Nature 2007; 3;447(7140):92-6.

 

 JS

-Jeffrey SoRelle, MD, is a 1st year Clinical Pathology Resident at UT Southwestern Medical Center.

Erin McElvania TeKippe, PhD, D(ABMM), is the Director of Clinical Microbiology at Children’s Medical Center in Dallas Texas and an Assistant Professor of Pathology and Pediatrics at University of Texas Southwestern Medical Center.

Massively Parallel – the Next Generation of Sequencing

Sounds like a good title for a sci-fi novel, right?  What is the big deal about Next Generation Sequencing (NGS)?  Otherwise known as massively parallel sequencing or high throughput sequencing, NGS has become a technique used by many molecular labs to interrogate multiple areas of the genome in a short amount of time with high confidence in the results.  Throughout the next few blogs, we’ll discuss why NGS has become the next big thing in the world of molecular.  We’ll go through the steps of setting up the specimens to prepare them to be sequenced (library preparation), what types of platforms are available and what technologies they use to sequence.  Lastly, we’ll go through some of the challenges with this type of technology.

Let’s start with a review of dideoxy sequencing, otherwise known as Sanger sequencing, which has been the gold standard since its inception in 1977.  A typical setup in our lab for this assay begins with a standard PCR to amplify a region of the genome that we are interested in, say PIK3CA exon 21, specifically amino acid 1047, a histidine (CAT).  The setup would include primers complementary to an area around exon 21, a 10x buffer, MgCl2, a deoxynucleotide mix (dNTP’s), and Taq polymerase.  After amplification, the resulting products would be purified with exonuclease and shrimp alkaline phosphatase (SAP).  Next, another PCR would be set up using the purified products as the sample and using a similar mix as in the original amp, but with the addition of a low concentration of fluorescently labeled dideoxynucleotides.  These bases have no -OH group, so when they are incorporated into the product, amplification ceases on that strand.  Because they are present in a lower concentration, the incorporation of these is random and will occur at each base in the strand eventually.  The resulting products are then run and analyzed on a capillary electrophoresis instrument that will detect the fluorescent label on the dideoxynucleotides at the end of each fragment.  Shown below is an example of the output of the data:

NGS1

The bases will be shown as peaks as they are read across the laser.  The base in question in the middle of the picture is, in a “normal” sequence, an adenine (A), as seen in green.  In this case, there is also a thymine (T) detected at that same location, as seen in red.  This indicates that some of the DNA in this tumor sample has mutated from an A to a T at this location.  This causes a change from a histidine amino acid to a leucine (p.His1047Leu) and is a common mutation in colorectal cancers.

So all of this looks great, right?  Why do we need to have another method since we have been using this one for so long and it works so well?  There are a few reasons:

  1. The sensitivity of dideoxy sequencing is only about 20%.  This means lower level mutations could be missed.  The sensitivity of NGS can get down to 5% or even lower in some instances.
  2. The above picture shows the sequencing in the forward direction as well as the reverse direction.  This area then has 2x coverage – we can see the mutation in both reads.  If we could get a higher coverage of this area and be able to sequence it multiple times and see that data, we could feel more confident that this mutation is real.  In our lab, we require each area has 500x coverage so that we feel sure that we have not missed anything.  The picture below displays the same sequenced area as in the dideoxy sequencing above.  This a typical readout from an NGS assay and, as you can see, this base has a total of 4192 reads, so it has been sequenced over four thousand times.  In 1195 of those reads, a T was detected, not an A.  We can feel very confident in these results due to how many times the area was covered.
  3. The steps above detailed only amplifying this one area, but with colorectal cancer specimens, we want to know the status of the KRAS, BRAF, NRAS, and HRAS genes as well as other exons in PIK3CA  Using the dideoxy sequencing method is a lot of time and effort.  NGS can cover these areas in these five genes as well as multiple other areas (our assay looks at 207 areas total) all in the same workflow

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Join me for the next installment to discover the first steps in NGS workflow!

 

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-Sharleen Rapp, BS, MB (ASCP)CM is a Molecular Diagnostics Coordinator in the Molecular Diagnostics Laboratory at Nebraska Medicine.