Essential Diagnostics List

A propos of Lab Week 2018, the WHO announced the development of an Essential Diagnostic List (EDL). The first Strategic Advisory Group of Experts on In Vitro Diagnostics (SAGE IVD) met in Geneva in April. The role of the SAGE-IVD is to act as an advisory body to matters of global policy and strategies related to in vitro diagnostics (IVDs) – to guide the development of the EDL.

The EDL is, as it sounds, a catalog of IVDs that are essential for diagnosis, treatment, and management of diseases. An EDL was called for in 2016 by Dr. Tim Amukele, a clinical pathologist at Johns Hopkins and President of the non-profit organization Pathologists Oveseas, and Dr. Lee Schroeder, a clinical pathologist at University of Michigan Ann Arbor (N Engl J Med 2016; 374:2511-4). Amukele and Schroeder suggested the EDL to complement the WHO’s Essential Medicines List (EML). They suggested 19 categories of IVDs that are essential for 10 of the medicines appearing on the EML. As it stands, the initial EDL focuses on 4 disease areas: HIV, TB, Malaria, and Hepatitis B & C. The following categories are provided for each disease area: analyte, intended use, level of facility that should have the IVD, assay format, specimen type, and links to WHO guidelines and any WHO prequalified or endorsed products.

For example, in the disease area “Malaria”, the analyte P. falciparum has the intended use of diagnosis of P. falciparum. The rapid diagnostic assay format is recommended for all level facilities. The specimen type is capillary whole blood, and the corresponding WHO guideline is “Good practices for selecting and procuring rapid diagnostic tests for malaria, 2011”.

The EDL will provide countries a way to focus attention on which tests are most appropriate, which can have a huge impact on the cost-effectiveness of the health care system, and also improve the quality of the laboratory results. Cost-effectiveness can be achieved by 1) focusing on evidence-based IVDs appropriate for a specific disease burden and 2) facilitating proper utilization of medicines and other clinical supplies necessary for treatment/management. In my experience, many laboratories in resource-limited areas are developed seemingly on a whim; testing might reflect a pet project of an absentee lab director, or donated equipment. Focusing on more appropriate testing, as Dr. Amukele told Clinical Laboratory News, give a lab more bang for their buck. Dr. Schroeder indicated that “lab testing develop ad hoc is more prone to quality issues”. The hope is that providing more direction for development of lab testing will encourage greater quality control programs. One way I can see that playing out is, if more labs in a specific area adopt the same testing, a shared sample program for cost-effective proficiency testing might be developed.

The WHO hopes that countries will use the EDL to develop country-specific EDLs, based on the disease burden specific to the country. National EMLs have been successful. Personally, I am very excited about the EDL! The WHO efforts to control HIV, Malaria, and TB have highlighted the need for laboratory diagnostics. I think it’s about time that labs got a chance to show their worth! The EDL is an important step in bringing the lab out of the basement and onto the global health stage.

 

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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.  

Why is it Important to Learn About Generations?

Understanding and appreciating different generations is critical for effective and productive teams, departments, and companies. Currently, there are five different generations in the workplace: Traditionalists, Baby Boomers, Generation X, Generation Y/Millennials, and Generation Z. A wide variety of experiences exist between these generations. For example, most traditionalists grew up without television, while almost all Generation Z’ers have a cell phone. If we look deeper, however, we can see commonalities between Traditionalists and Gen Z; both grew up during economic strife (The Great Depression and the Great Recession, respectively). Understanding each other’s views and values will allow different generations to increase their appreciation of one another. This, in turn, will lead to better communication and collaboration because people are now talking from a sense of appreciation and acknowledgement. When people feel heard, understood, and valued, they are more likely to invest time and energy into their projects and jobs and they are more likely to stay at an organization. Truth is, we need people of all generations to make organizations effective. You want the “getting the job done” attitude of the Traditionalists, the teamwork skills of Baby Boomers, the self-reliance of X’ers, the multitasking abilities of Millennials, and the entrepreneurship of Generation Z. Combined, these qualities create a powerful workforce that is able to handle any challenge that comes its way.

It is important to remember that learning can, and should, go both ways: newer generations can pay attention to the older generation’s lessons and knowledge, while older generations can learn a lot from the younger ones (and not just about how to use technology). Each generation has its own unique perspective, challenges, and contributions, and we can all grow by listening to and learning from people who are different than us. Generational diversity is one way to strengthen your team.

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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.


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The differences are many and yet so few.  This is stated so clearly by Gretchen Gavett when she wrote in the Wall Street Journal:

“Baby Boomers, Gen Xers, Millennials, the Gen Z up-and-comers – we all want the same things, (income, sure, but also purpose, and to feel valued) just in slightly different ways. The challenge is to look past the stereotypes and listen to one another so that good work gets done efficiently and humanely.”        

Let’s begin with the GI Generation. The youngest of this generation are in their early 90’s so they are almost non-existent in the workplace.  They are our oldest living generation and were born at the beginning of the 19th century. Most of the soldiers during WWII came from this generation.

Traditionalists make up 2% of the current workforce which is the smallest percentage. However, they represent the institutional memory of a workplace. They know and remember the organization’s past and founding goals. Typically born between 1927 and 1945, they went through their formative years during the Great Depression and its aftermath.

Baby Boomers are currently the largest generation at approximately 77 million people in the United States. (Generation Y runs a close second.) Born between the years of 1946 and 1964, they are the post-World War II generation. The Baby Boomers represent about 29% of the workforce; that number is declining by the day.

Generation X is bookended by the two largest generations, Baby Boomers and Generation Y. They are born between 1965 and 1980. They make up approximately 23% of the workforce.

Generation Y, also known as the Millenials, are born between 1981 and 2000. The Millenials are currently about 42% of the workforce, which makes them the largest working generation.  They have their own values and characteristics (as do the other generations) their numbers make them a force to be reckoned with. 

Generation Z is our newest generation.  They’re currently around 4% of the workforce and growing.  They grew up during the great recession after the early 2000’s.  We are learning about what the Generation Z’s value and their characteristics as each day passes.

The challenge we all face: how can we connect, communicate, and collaborate most effectively in the workplace and outside of the workplace?

Source: https://hbr.org/2009/10/are-you-ready-to-manage-five-g

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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.  

 

Hematopathology Case Study: A 45 Year Old Male with Mediastinal Mass

Case History

A 45 year old male underwent a chest MRA for aortic dilation due to his history of an aneurysmal aortic root. Upon imaging, an incidental anterior mediastinal mass was seen that measured 4.0 cm. In preparation for an upcoming cardiac surgery, the patient underwent a thymectomy with resection of the mass. The sample is a section from the mediastinal mass.

Diagnosis

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H&E, 2x
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H&E, 10x. Green Arrows: “lollipop” germinal centers
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H&E, 10x. Red arrow: focal “twinning” of germinal centers

Sections show an enlarged lymph node with several follicles demonstrating atrophic-appearing germinal centers which are primarily composed of follicular dendritic cells. These areas are surrounded by expanded concentrically arranged mantle zones. Focal “twinning” of germinal centers is present. Additionally, prominent centrally placed hyalinized vessels are seen within the atrophic germinal centers giving rise to the “lollipop” appearance.

By immunohistochemistry, CD20 highlights B-cell rich follicles while CD3 and CD5 highlight abundant T-cells in the paracortical areas. CD10 is positive in the germinal centers while BCL2 is negative. CD21 highlights expanded follicular dendritic meshwork. CD138 is positive in a small population of plasma cells and are polytypic by kappa and lambda immunostaining. HHV8 is negative. MIB1 proliferation index is low while appropriately high in the reactive germinal centers.

Overall, taking the histologic and immunophenotypic findings together, the findings are in keeping with Castleman’s disease, hyaline vascular type. The reported clinical and radiographic reports suggest a unicentric variant.

Discussion

Castleman’s disease comes primarily in two varieties: localized or multicentric. The localized type is often classified as the hyaline vascular type (HVCD). Demographically, it’s a disease of young adults but can be found in many ages. The most common sites for involvement are the mediastinal and cervical lymph nodes.

The classic histologic findings of HVCD involve numerous regressed germinal centers with expanded mantle zones and a hypervascular interfollicular region. The germinal centers are predominantly follicular dendritic cells and endothelial cells. The mantle zone gives a concentric appearance, often being likened to an “onion skin” pattern. Blood vessels from the interfollicular area penetrate into the germinal center at right angles, giving rise to another food related identifier, “lollipop” follicles. A useful diagnostic tool is the presence of more than one germinal center within a single mantle zone.

The differential diagnosis of HVCD includes late stage HIV-associated lymphadenopathy, early stages AITL, follicular lymphoma, mantle cell lymphoma, and a nonspecific reactive lymphadenopathy. A history of HIV or diagnostic laboratory testing for HIV would exclude the first diagnosis. AITL usually presents histologically as a diffuse process but atypia in T-cells with clear cytoplasm that co-express CD10 and PD-1 outside of the germinal center are invariably present. EBER staining may reveal EBV positive B immunoblasts in early AITL, which would be absent in HVCD. The most challenging differential would include the mantle zone pattern of mantle cell lymphoma. Flow cytometry revealing a monotypic process with co-expression of cyclin D1 on IHC would further clarify the diagnosis.1

Overall, unicentric Castleman’s disease is usually of the hyaline vascular type. Surgical resection is usually curative in these cases with an excellent prognosis.2

 

References

  1. Jaffe, ES, Harris, NL, Vardiman, J, Campo, E, Arber, D. Hematopathology. Philadelphia: Elsevier Saunders, 2011. 1st ed.
  2. Ye, B, Gao, SG, Li, W et al. A retrospective study of unicentric and multicentric Castleman’s disease: a report of 52 patients. Med Oncol (2010) 27: 1171.

 

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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.

Lymphocytosis Can Be Anything

Case History

A 63 year old patient presented with a high white cell count of 108 K/uL and thrombocytopenia of 110 K/uL.

Peripheral smear examination revealed marked lymphocytosis with presence of numerous small to medium sized lymphoid cells with round to oval nuclei, clumped nuclear chromatin and variable amount of cytoplasm, some with cytoplasmic projections. As the features were consistent with a lymphoproliferative disorder peripheral blood was sent for flow cytometry.

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Based on the morphology the differential diagnosis included B-cell lymphoproliferative disorders such as marginal zone lymphoma, hairy cell leukemia/variant, or less likely chronic lymphocytic leukemia and/or mantle cell lymphoma.

Flow cytometry revealed presence of clonal B-cells expressing CD19, CD20, Cd11c, CD103 and FMC-7. The cells were negative for CD5, CD10, and CD25.

The phenotype together with the morphology and CBC findings were diagnostic of hairy cell leukemia variant.

Discussion

Hairy cell leukemia variant ( HCL-v) is a B-cell lymphoproliferative disorder that resembles classic hairy cell leukemia but exhibits variant cytological and hematological features such as leukocytosis and also shows variant immunophenotype including absence of CD25, CD123 and/or annexin A1.

HCL-v is about one tenth as common as HCL (hairy cell leukemia) with an annual incidence of approximately 0.03 cases per 100,000 population. There is slight male preponderance. Patients with HCL-v typically present with leukocytosis with an average WBC of 30 K/ul and /or thrombocytopenia.

The 5 year survival rate is around 50-60%. Most patients require therapy which can range from splenectomy to combination chemotherapy with Rituximab.

 

Reference

  1. WHO classification of Tumors of Haematopoietic and Lymphoid Tissues; IARC 2017

 

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-Neerja Vajpayee, MD, is the director of Clinical Pathology at Oneida Health Center in Oneida, New York and is actively involved in signing out surgical pathology and cytology cases in a community setting. Previously, she was on the faculty at SUNY Upstate for several years ( 2002-2016) where she was involved in diagnostic work and medical student/resident teaching.

Library Preparation – The First Step in a NGS Setup

Welcome back! Last quarter we discussed why Next Generation, or Massively Parallel, Sequencing is the next big thing in the world of Molecular Diagnostics. The sensitivity, the depth of coverage and the ability to interrogate many different areas of the genome at the same time were just a few of the benefits of these types of assays. Next, I would like to describe a couple different methods of library preparation, which is the first step necessary to run an NGS assay.

First of all, let’s define “Library.” I find this is the most common question technologists new to this technology ask. Essentially, a library is a specimen’s collection of amplicons produced by the assay that have been barcoded, tagged with appropriate platform adapters and purified. These will serve as the input for the next part of the NGS workflow, clonal amplification (the topic of next quarter’s blog!).  How these libraries are prepared differ depending on platform (i.e, Ion Torrent vs. MiSeq), starting material (RNA vs. DNA), and type of assay (targeted amplicon vs. exome).

Before we begin the library prep discussion, a note about the input specimen. The DNA must be quantitated using a method that is more specific than spectrophotometry – it must be specific for double-stranded DNA. It will lead to an overestimation of the amount of DNA in the specimen, which will lead to over-dilution and consequently, lower quantity of final library. Real-time PCR and a double-stranded kit with fluorometry are two examples of assays that will give accurate concentrations of double-stranded DNA.

Our lab has begun using NGS for some of our oncology assays, so I will focus on the two types we perform currently, but keep in mind, there are many other types of assays and platforms.

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Image 1: ion torrent amplicon library preparation. Source: Ion AmpliSeqTM Library Preparation User Guide – MAN0006735, Rev. 10 September 2012.

The assay we use for our Ion Torrent platform is a PCR amplicon based assay. The first step is to amplify up the 207 regions over 50 genes that contain hotspots areas for a number of different cancer types. This all occurs in one well for each specimen. Once those areas are amplified, the next step is to partially digest the primer sequences in order to prepare the ends of amplicons for the adapters necessary for the sequencing step. As shown in the figure above, two different combinations of adapters may be used. The top one, listed as the A adapter (red) and the P1 adapter (green), would be used if only one specimen was to be sequenced on the run. The A and P1 adapters provide universal priming sites so that every amplicon of every sample can be primed with the same primers, rather than having to use gene specific primers each time. The second possibility is listed below that, with the same P1 adapter (green) and a Barcode Adapter labeled X (red and blue) – it still contains the A adapter necessary for sequencing (red), but it also contains a short oligonucleotide sequence called a “barcode” (blue) that will be recognized during the analysis step based on the sequence. For example, Barcode 101’s sequence is CTAAGGTAAC – this will be assigned to specimen 1 in the run and all of the amplicons for that specimen will be tagged with this sequence. Specimen 2 will have the barcode 102 (TAAGGAGAAC) tag on all of its amplicons. During analysis, the barcodes will be identified and all of the reads with the 101 sequence will be binned together and all of the reads with the 102 sequence will be binned together. This allows many specimens to be run at the same time, thus increasing the efficiency of NGS even more. Lastly, the tagged amplicons are purified and normalized to the same concentration.

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Image 2: MiSeq amplicon library preparation. Image source: https://www.illumina.com/content/dam/illumina-marketing/documents/applications/ngs-library-prep/for-all-you-seq-dna.pdf

The assay we use for our MiSeq platform is a hybridization followed by PCR amplicon based assay. The first step is to hybridize probes to 568 regions over 54 genes that contain hotspots for a number of different cancer types. This occurs in one well for each specimen. Once the probes have hybridized, the unbound probes are washed away using a size selection filter plate. Next, the area between the probes is extended and ligated so that each of the 568 amplicons are created. These are then amplified in a PCR step using primers that are complimentary to a universal priming site on the probes, but also contain adapters plus the two indices required for paired end sequencing (the Ion Torrent platform utilizes single-end sequencing – this will be discussed in the sequencing portion in an upcoming blog!). As in the previous method, after PCR, these tagged amplicons are purified and normalized to the same concentration in preparation for the next step – clonal amplification.

Stay tuned for next quarter’s post – clonal amplification!

 

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