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.

Poll Friday: Thalassemia

 

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. 

Friday Poll: Variable K+ Results

Here’s to Fresh STARTs: Sustainable Transitions And Reachable Targets

From the title, you might be able to tell that I’ve been busy on Lab Management University (LMU). Going through the online modules and lessons in the LMU certificate program I mentioned this past May, I’ve been able to hone several skills in interpersonal communication, resource management, and project leadership. (A worthwhile investment through ASCP which I highly recommend!) Another thing that’s kept me quite busy over the last two years has been Zika-virus and mosquito-related public health initiatives both inside and outside the laboratory. In a recent blog post, I discussed the correlation between measuring success in projects like these just as one would with common lab-centric goals. And, as a conclusion to that hard work, this will be my last directly-related Zika/public health post. Transitioning to the second half of medical school, I’ll be leaving behind a true grassroots project that not only reached countless people but has the promise to be sustainable after my departure from the island of Sint Maarten back to the states.

As with many times in life, I would say fresh starts are a welcome chance to reflect and grow upon things you might have learned or goals you might be closer to finishing. What has been made clear to me in my time working through classrooms, cases, exams, and projects is that the “jargon” we use as laboratorians is definitely worth its weight. It isn’t full of hollow charges for metrics and goals; it’s about real data and real solutions. Having the ability to apply my prior experiences in laboratory medicine throughout medical school—both inside and outside the classroom—has been an invaluable benefit. The general principles that guided my last blog post reflected simple goals (i.e. turnaround time compared with public health metrics) which utilize fundamental models of data collection, adjustment, and success. The essential model I described a few months ago is now a mainstay of a project that will continue to improve public health statistics slowly as time goes on.

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The model as it stands now consists of clear steps to identify problems which require interventions, highlight gaps in current practice, data collection from literature exists currently, collaboration with partners in a community of trust, and continuing those partnerships as improvements are made incrementally over time. The model has been repeated and successfully modified for these last two years from on-campus blood testing with procedural write-ups and data evaluation, to teaching school-aged children about mosquito prevention, to partnering with local government officials and having your projects adopted into their portfolio, and visiting individuals in their homes to discuss health and prevention.

To keep it brief, I’ve had an amazing experience here being able to lead and contribute to a wonderful and impactful project such as this. It has become increasingly clearer to me throughout this work that the values and skills programs like LMU teach are directly parallel with successes in various clinical settings. From the bedside to the laboratory to public health in the field, the lessons of how to effectively engage people and solve problems are critical. My time in laboratories before medical school, the daily grind of classwork here, and the projects I’ve been able to write about have all given me the space to try these skills on real situations—and hopefully that will make me the best clinician I can be after medical school is completed. Check out my “highlight reel” of partnerships, workshops, and active management in Zika prevention below.

Be sure to check back here next time, I’ll be writing from my hometown as I’ll explore ASCP’s Annual Meeting in Chicago, IL this coming September and report back on why it’s important to network and stay involved with our great professional community. Thanks for reading!

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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.