New Year. New Skills.

I do not recall if it was an email or if I saw it on the ASCP website, but the byline caught my attention: New Year. New Skills. My mind quickly started racing. January marks a fresh beginning, the time to make new resolutions, the time to feel the excitement of new possibilities. 

The Issue

We are more than halfway through the month and I have yet to identify the skill I would next like to acquire. So many questions! So much to learn, so little time! How do you choose what to focus on? Where do you start? What can you manage? Is there anyone who can help or teach you? And if you are like me, you might also ask yourself, “Why do I always pile more on my plate?” Maybe this is the year you choose to learn to say no? Nah. So what’s it going to be?

The Solution

Since our lives are all different and there are millions of possible distinct scenarios, I will share what I decided to do. First, I evaluated my work-life balance and determined if I wanted to acquire a skill that would benefit my work (career and ambition) or lifestyle (health, pleasure, leisure, family) (1). I also took into consideration how much more I could fit onto my already overflowing plate.

I decided to work on something that would help me with both work and lifestyle (because who doesn’t like to maximize their return on investment?). I chose something I do not like to do, something that scares me, something I have difficulty with, something I avoid like the plague, but most importantly it’s something that I wish I could do better; a skill that I envy: having difficult conversations.

Communication is a vital component of our lives. We all communicate, but how many of us have mastered the skill of communicating? Also, there are many aspects of communication (2). Poor communication can make or break a situation or relationship. Being able to communicate well is a great skill to possess (3). Reference two provides a long list of skills that I highly recommend you also take a look at ( I went down the list and individually assessed which skills I feel that I do well with and which ones I do not (2). This little exercise served as a reality check as to where I stand in regard with my aptitude to communicate. I invite you to do the same. You may be surprised at what you find!

The Importance of Good Communication

As a laboratory director, many facets of my job depend on my ability to communicate well. I must communicate with clinicians, technologists, administrators, other coworkers, vendors, students, etc. Not only do I communicate with a variety of groups of people, in a multitude of different platforms (individually, small groups and meetings, or large groups; such as national conferences), but it is also important that my written, verbal, and non-verbal communication skills are clear and easily understood.

As laboratory professionals, one very important aspect of our job is to communicate critical results. It is essential that we not only relay the data, but it is equally important for us to communicate it well so that the clinician completely understands the information so that they can properly care for the patient. Moreover, we must not forget the golden rule: garbage in, garbage out. What I mean by this is that good communication should begin in the pre-analytical phase. We want the clinician to provide the laboratory with the best possible specimen so that in turn, we can provide them with the most accurate result. So how do we ensure that we obtain the best possible specimen? We communicate.

The laboratory communicates our needs to the provider in order to properly do our job. For example, we provide detailed information on how to properly collect specimens, which container type to use, how to handle the specimen, how much (volume) specimen to submit, which temperature to submit the specimen, etc. Properly communicating these details is essential.

The Difficult Conversation

As laboratory professionals, we are just one part of a larger healthcare team. If you stop to think about it, we all have to participate in difficult conversations as part of our jobs. Doctors have to tell patients that they are going to die, laboratory professionals have to tell clinicians we lost their specimen, executive administrators have to tell downstream leadership that the budget has been cut again, managers and supervisors have to tell employees they are being written up or worse. Being able to successfully have a difficult conversation would serve us all well. As such, most institutions provide classes or webinars to help employees develop this skill.

The definition of difficult is: not easily or readily done; requiring much labor, skill, or planning to be performed successfully; hard (4). Carrying out a difficult conversation with grace is an extraordinary skill that encompasses a variety of communication attributes. Regardless of the scenario, the communicator must be clear, articulate, and courteous. However, depending on the scenario, being concise, confident, strategic, diplomatic, convincing, empathetic, motivating, open-minded, and/or quick thinking may also be useful skills to possess during a difficult conversation. Other valuable skills are conflict management, being able to explain, and/or listening. 

The Conclusion

For many, the New Year marks the time to set new goals, to accept new challenges, and welcome new beginnings. Why not use this opportunity to learn a new skill? The good news is that no matter what your new skill will be, it will also benefit your health. In order to acquire a new ability, you must work to actively learn to become proficient in that ability; therefore learning a new skill will also benefit your brain function. There are many studies that demonstrate that active learning keeps the mind sharp (5). Challenging your mind improves brain function and active learning slows cognitive decline (6). If you want to be brave, then don’t only choose a skill that will be fun or helpful, but choose to learn something that also challenges you to face one of your fears. For me, I hope to learn how to master the art of having difficult conversations….successfully. In the words of Marie Curie, “Nothing in life is to be feared, it is only to be understood. Now is the time to understand more, so that we may fear less.”

Happy learning! Happy New Year!


The References

  1. Work-life Balance.–life_balance. Accessed January 16, 2018.
  2. The balance. List of Communication Skills for Resumes. Accessed January 16, 2018.
  3. The balance. Communication Skills for Workplace Success. Accessed January, 16, 2018.
  4. com. Difficult. Accessed January 16, 2018.
  5. Stenger, M. 2013. New Study Shows How Active Learning Improve Cognitive Function. Accessed January 17, 2018.
  6. Park, D.C., Bischof, G.N. 2013. The aging mind: neuroplasticity in response to cognitive training. Dialogues Clin Neurosci. 15(1): 109-119. PMC23576894. Accessed January 17, 2018.


Martinez Headshot-small 2017

-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.

Organization Savvy

Understanding culture is essential to fit in anywhere: when you are traveling, with your family, and where you work. Understanding and appreciating culture is important, because you do not want to unintentionally offend someone by over-explaining a task, asking about personal lives when it is not considered appropriate, or going straight to business and skipping informalities completely when it is expected to get to know each other before attending to tasks at hand. Understanding organizational culture is therefore important for every employee. However, creating a fostering a productive culture is the responsibility of the leaders.

There is a critical distinction between the culture and the climate of an organization. Culture refers to how employees feel they are expected to do things and behave. Climate is the causal factors of the culture. Another way of putting it is that culture reflects the shared values, beliefs, norms, and expectations; climate reflects the outcomes of the culture such as engagement, teamwork, and perceived quality of work. However, it is important to note that there are two levels of culture: the ideal culture, or what should be expected; and the current culture, or what is actually expected. If we compare the climate to the human body, climate is the pulse, temperature, and blood pressure. Culture would be the bigger medical issues such as flu or cancer. The ideal culture would be a cancer-free body, while the current culture might be a body with a cancerous tumor caused by the climate of smoking. This example shows that culture is harder to change than climate: it is easier to change someone’s blood pressure than to change their cancer status.

Climate can therefore also drive and change the culture. If you want to change something in the culture, you can start with changing the climate. Let’s take the following organization as an example in which the ideal culture is that team work is valued, but the current culture shows that competitiveness is valued. The causal climate factors are in the structure of performance reviews: only individual and competitive behavior is analyzed, but not team behavior. If we would thus change the structure of the performance review (climate) we could change the current culture (competitiveness) closer to the ideal culture (teamwork).

The Organizational Savvy course focuses on how you see the current culture of your organization. It organizes the culture in three different clusters: constructive, passive/defensive, and aggressive/defensive. Each of these clusters are then divided into four styles, allowing you to understand your culture in more detail. The course explains how to improve the culture of the organization based on the profile.

Understanding what is expected in your organization sets you up for professional success and allows you to be an active member of establishing effective organizational culture. Whether you are a leader, chair, employee, or staff member, taking proactive steps to understand and foster organizational success will set you and the rest of your team and organization up for prosperity.


-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.


Organization Savvy is the core of high-performing organizations. It is a mixture of staff’s emotional intelligence, interpersonal relations, skills, self-awareness, competencies, and optimized organizational culture. With my experience in centralized laboratory services, improving quality in thirty laboratories with a wide variety of organizational cultures was a big challenge. To enhance the best practices and empower quality improvement projects, organizational savvy is required. The best tools to achieve this were creating an organizational influence map, building relationships with different laboratory staff, influencing people by turning the organization into learning atmosphere, and the most important, avoid negative players.

With my journey towards excellence, I had to communicate with different leadership styles. I had great outcomes with constructive style leaders, as they are task- and people-oriented, collaborative, achievers, humanistic, and encouraging. I had some difficult times with aggressive/defensive leaders who have some insecurities. I had to assure them that our journey towards excellence is to build people before building the organization. In addition, they had to realize our organizational objectives were in full alignment with their core values and career goals. I worked on improving my personal power by using self-awareness assessments and improving emotional intelligence. My skills and knowledge in Quality of Laboratory Medicine were good enough to build credibility inside the organization. My future goal is to increase my circle of influence and reduce my circle of concerns to achieve organizational savvy and hence, the organizational vision towards excellence.


Photo -Rana

-Dr. Rana Nabulsi has a PhD in Quality Management and a master’s degree in molecular genetics. She is currently the Head of Quality at Pathology and Genetics department at Dubai Health Authority and the Chair of Advisory Board at the American Society for Clinical Pathology (ASCP) in UAE. She is doing her fellow at the American Collage of Healthcare Executives (ACHE). She is a Certified Professional in Healthcare Quality (CPHQ) by the National Association of Healthcare Quality (NAHQ)-US and certified for Green Belt -Six Sigma (SSGB) by the American Society of Quality (ASQ). Dr. Nabulsi has experience in leading more than twenty medical laboratories in achieving the College of American Pathologist (CAP), ISO 15189, and American Association of Blood Bank (AABB) accreditations. She is certified as lead auditor for ISO 9001, ISO 15189, ISO17025, and OHSAS 18001 management systems. Dr. Nabulsi is a certified EFQM Assessor, trainer and public speaker for local and International Healthcare conferences about healthcare quality, safety, and leadership.


Microbiology Case Study: An 89 Year Old with an Infected Wound

Case History

An 89-year-old gentleman presented with a cough, subjective fever, and potential wound infection on his forehead. He has a past medical history notable for hyperlipidemia, coronary artery disease, chronic congestive heart failure, past STEMI, history of gastric lymphoma, diabetes, Parkinson disease, and stage III chronic kidney disease. Two months prior to presentation, he fell down the steps of his apartment and suffered a laceration of his forehead that ultimately required surgical repair. He was discharged home with wound care instructions. He was doing relatively well up to 5 days prior to presentation when he started to develop a small, soft, erythematous lesion on his forehead with swelling and non-purulent appearing serous drainage at the site of repair. After it persisted, he presented to the emergency department where he underwent a small incision and drainage. A specimen was collected in a sterile syringe and sent to the microbiology laboratory for culture.

Image 1. Gram stain from a fluid culture illustrating filamentous branching gram-positive bacilli (100x, oil immersion).
Image 2. Modified Kinyoun stain from a fluid culture illustrating filamentous branching modified acid fast bacilli (100x, oil immersion).
Image 3. Chocolate agar illustrating chalky white colonies.

The organism was confirmed as Nocardia abscessus/asitica by a reference laboratory.


Nocardia is a genus of aerobic, weakly Gram-positive, catalase-positive, rod shaped bacteria that are modified acid-fast and form beaded branching filaments. They grow slowly on commonly used nonselective culture media, with colonies becoming evident in 3-5 days, and have the ability to grow in a wide temperature range. Colony morphology is variable, with colors ranging from white to orange and texture ranging from smooth to chalky. They are saprophytic organisms that are commonly found in soil, organic matter, and in the oropharynx as normal flora. Virulence factors for Nocardia include catalase, superoxide dismutase, and cord factor. Cord factor prevents lysosomal fusion with the phagosome, thus inhibiting phagocytosis by macrophages.

There are more than 80 species of Nocardia causing various forms of human disease, the most pathogenic of which are: N. asteroides, N.braziliensis, N.caviae, N.nova and N.abscesses. Symptoms can range from a localized lung or cutaneous infection to disseminated disease. Most infections involve the lung initially following inhalation of the organisms which commonly spread to extrapulmonary sites with the disease being more severe and likely to disseminate in immunosuppressed patients. In addition, the skin can be a site of primary infection through traumatic inoculation in immunocompetent patients.

Primary cutaneous infections occur in 5% of cases and manifest in one of the following ways: lymphocutaneous infection, mycetoma, superficial cellulitis, or localized abscesses. The most commonly involved sites for cutaneous infections are the extremities, though infection can affect any area, including the head and neck.

Treatment for primary cutaneous nocardiosis includes antimicrobial therapy and surgical irrigation and drainage when appropriate. Though antibiotic therapy is recommended, spontaneous resolution can occur without treatment. Trimethoprim-sulfamethoxazole is used most frequently for nocardiosis with the usual duration of therapy being 2-3 months in those cutaneous disease.


  1. Wilson JW. Nocardiosis: Updates and Clinical Overview. Mayo Clinic Proceedings. 2012;87(4):403-407. doi:10.1016/j.mayocp.2011.11.016.
  2. Vijay Kumar GS, Mahale RP, Rajeshwari KG, Rajani R, Shankaregowda R. Primary facial cutaneous nocardiosis in a HIV patient and review of cutaneous nocardiosis in India. Indian Journal of Sexually Transmitted Diseases. 2011;32(1):40-43. doi:10.4103/0253-7184.81254.
  3. Lee TG, Jin WJ, Jeong WS, et al. Primary Cutaneous Nocardiosis Caused by Nocardia takedensis. Annals of Dermatology. 2017;29(4):471-475. doi:10.5021/ad.2017.29.4.471.
  4. Outhred, A.C., Watts, M.R., Chen, S.CA. et al. Nocardia Infections of the Face and Neck. Curr Infect Dis Rep 2011 Apr;13(2):132-40. doi: 10.1007/s11908-011-0165-0.


-Clayton LaValley, MD is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.


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


Safety Motivation

If you search for top motivational movie speeches, you will see things that might work in real life. The President’s speech from Independence Day (1996), for example, might influence you to never be oppressed by alien tyranny. Freedom will be your rally cry after listening to William Wallace in Braveheart (1995), or Maximus from Gladiator (2000) can speak to your heart about teamwork. Unfortunately, such speeches do to tend to maintain motivation for great lengths of time. Also, none of them will translate to a motivational discussion about safety with your lab staff.

Over many years I have watched what motivates people to do the right thing or take the safe actions in the laboratory, and that motivation varies. Different groups of people are persuaded by different forces, and understanding that can help you move your lab safety culture in the direction you desire. You may not agree with or even like some of the influencers, but learning them can help you be more effective in achieving overall safety compliance.

They say money is a motivator for people in all kinds of circumstances, and that’s true for lab safety as well, although not in the way you might believe (while some businesses may pay a bonus for fewer safety incidents, that is not typical in the lab setting). Lab staff who are concerned about finances are more open to following some lab safety practices if they realize the cost savings. Obviously, lab injuries and exposures cost the department both monetarily and with staff absences. Following proper regulations can reduce costly citations and fines that can be levied by organizations like OSHA, the EPA, or CMS. Some lab team members want funds available for new equipment or more staff. Use that to encourage them to follow proper safety procedures. Make sure staff properly segregates waste in the lab, for example, since doing things like placing paper into a sharps container costs the department extra money. Hospital and lab leadership also respond well to financial motivation. If you need something fixed or replaced because it is unsafe, always explain the financial consequences to the facility if the fix is not approved.

Knowledge can also be a powerful safety stimulator for some staff. Understanding the consequences of poor safety behaviors will discourage some, and education about those consequences needs to be given regularly. Let’s look at waste disposal again- those who are concerned about the environment should know that tossing clean items into a biohazard container could increase the need for biohazard landfills in the area- something we should avoid. Talking about the follow up testing and unpleasant effects of prophylaxis following an exposure from an unknown source can be very eye-opening. It may spur staff to be more careful when potential exposure situations arise.

You might not like to hear that punishment can be a motivator for correct behaviors, but for some staff members it is. Sometimes, explaining that a written corrective counseling or even termination will occur if safety practices are not followed will keep laboratorians working carefully and correctly. No one wants to “threaten” people to do the right things, but there will be those who are only motivated by not wanting to “get in trouble.” Knowing who those employees are can be important to guiding your leadership approach when working with them.

Lastly, some lab staff are inspired to act safely because the environment is designed to make doing so easy. PPE is readily available- lab coats of all sizes are accessible, gloves are out and not in a drawer, and face protection is mounted conveniently. There are hooks for lab coats near exit doors and hand washing sinks so that staff can properly doff and exit. Cleaning supplies and spill kits are readily available and instructions to use them are posted and up to date. Warning signs are there for staff and for visitors not used to the dangers in the department. I know that many labs are older, and the physical layout is not always conducive to making safety easy, but there are always steps that can be taken in order to make safety easier to achieve. You may need to step back and look at your environment with fresh eyes in order to envision what can be done to make improvements.

Think about what incentives are important to you when it comes to lab safety. Is it simply self-preservation? That’s good, but for many who are complacent about safety, their motivation may be different. Finding their reasons to be safe is a worthwhile task. It helps you understand better who your staff is as a people, and it will help you gain expertise for providing the stimuli they need to continue to work safely today and every day.


Scungio 1

Dan Scungio, MT(ASCP), SLS, CQA (ASQ) has over 25 years experience as a certified medical technologist. Today he is the Laboratory Safety Officer for Sentara Healthcare, a system of seven hospitals and over 20 laboratories and draw sites in the Tidewater area of Virginia. He is also known as Dan the Lab Safety Man, a lab safety consultant, educator, and trainer.

Hematopathology Case Study: An 18-year-old Man with Acanthocytosis

Case History

We were asked to review the peripheral blood smear of an 18-year-old male who had presented to the emergency department with shortness of breath and abdominal distension. His past medical history was significant for numerous hospitalizations for recurrent fungal and bacterial pneumonia, pulmonary abscesses, osteomyelitis, necrotizing granulomas, and cervical lymphadenopathy requiring multiple lymphadenectomies. This history dates back to when he was 3 months old.

Blood Smear findings

The CBC demonstrated severe anemia and mild leukopenia. The peripheral blood smear showed numerous acanthocytes and poikilocytosis shown below.



Additional Clinical Findings

Abdominal ultrasonography demonstrated hepatosplenomegaly with enlarged porta-hepatis lymph nodes. Additionally, chest CT scanning demonstrated bilateral mass-like consolidations, prominent hilar lymphadenopathy, and osteolytic lesions of the vertebral bodies. A comprehensive investigation for opportunistic infections was negative. Lung and vertebral body biopsies (not pictured here) revealed poorly formed granulomas. A blood transfusion was considered; however, the patient had previously been demonstrated to express anti-Kx antibodies, which would require transfusion with exceedingly rare blood products.


The preceding case history describes a patient with a contiguous gene deletion syndrome that includes chronic granulomatous disease (CGD) and the McLeod phenotype, demonstrating a fascinating disorder with important implications in hematopathology and several other disciplines of pathology.


McLeod syndrome is a rare, X-linked disorder characterized by the deletion of the XK gene which encodes for the Xk protein. Overall, the lack of synthesis of the Xk protein leads to the lack of expression of the Kx antigen which in turn leads to a marked decrease in the quantities of Kell antigens. In this case, due to the presence of an anti-Kx antibody, the patient would require transfusion with either Kell-null or McLeod phenotype blood products. Unfortunately, only one unit of compatible blood was identified when the rare blood donor database was queried. The clinical team therefore elected for treatment with erythropoietin and iron supplementation which eventually lead to a modest increase in the patient’s hemoglobin concentration.

Acanthocytes, or spur cells, are spiculated red cells with a few projections of varying size and surface distribution that can be seen in a variety of clinical conditions including CGD with McLeod red cell phenotype. Other conditions include (but are not limited to) neuroacanthocytosis, malnutrition states, infantile pyknocytosis, (Lu) null Lutheran phenotype, hypothyroidism, myxedema, and Zieve syndrome. Acanthocytes should be distinguished from echinocytes, or burr cells, that also demonstrate multiple small projections but these are uniformly distributed on the red cell surface.

The prominent acanthocytosis seen in McLeod syndrome is thought to be due to an imbalance of the number of lipids in the inner layer relative to the outer layer. Related to this phenomenon is McLeod neuroacanthocytosis syndrome, a disorder with neurologic manifestations including movement disorders, cognitive alterations, and psychiatric symptoms. Although our patient did not exhibit these symptoms, McLeod neuroacanthocytosis syndrome is known to start in early to middle adulthood and the patient will need to be monitored for the onset of neurologic sequelae.

The McLeod phenotype is frequently associated with CGD due to the proximity of the XK gene to the CYBB gene on the X chromosome. The CYBB gene encodes for a subunit of the NADPH oxidase enzyme complex. A deficiency in NADPH oxidase activity leads to the characteristic increased susceptibility to severe bacterial and fungal infections seen in CGD. The nitroblue-tetrazolium test can be used to evaluate NADPH oxidase activity in the white blood cells and can help make a diagnosis of CGD. Histologically, CGD can show prominent necrotizing and non-necrotizing granulomas in various locations throughout the body.

Overall, treatment of CGD with McLeod red cell phenotype is supportive. There is no known cure or definitive treatment. The patient will likely continue to have infections with opportunistic organisms which will be treated on a case by case basis.


  1. Heyworth PG, Cross AR, Curnutte JT. Chronic granulomatous disease. Current opinion in immunology. 2003 Oct 31;15(5):578-84.
  1. Jung HH, Danek A, Walker RH, Frey BM, Gassner C. McLeod neuroacanthocytosis syndrome.
  1. Khodadad JK, Weinstein RS, Marsh LW, Steck TL. Shape determinants of McLeod acanthocytes. Journal of Membrane Biology. 1989 Mar 1;107(3):213-8.
  1. Watkins CE, Litchfield J, Song E, Jaishankar GB, Misra N, Holla N, Duffourc M, Krishnaswamy G. Chronic granulomatous disease, the McLeod phenotype and the contiguous gene deletion syndrome-a review. Clinical and Molecular Allergy. 2011 Nov 23;9(1):13.



-Michael Moravek, MD is a 2nd year anatomic and clinical pathology resident at Loyola University Medical Center. Follow Dr. Moravek on twitter @MoravekMD


-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.

Template Preparation, Clonal Amplification, and Cluster Generation – Oh My! – Step Two in an NGS Setup

Hello again – let’s continue our discussion of Next Generation, or Massively Parallel, Sequencing and how it is performed.  Over the last two blogs we have seen why NGS is being used in a Molecular Diagnostics Lab and how library preparation is executed.  Specifically, we reviewed how Ion Torrent and MiSeq libraries may be prepared for DNA amplicon sequencing.  The final product of this work is a collection of amplicons that have been amplified, barcoded, tagged with the appropriate platform adapters and purified.  These are what compose a specimen’s “library.”

The next step in NGS preparation is template preparation.  The main goal of this step is to create multiple copies of the same amplicon in close proximity so that when it is sequenced, it creates a strong enough signal to be detected.  This occurs for each amplicon in the specimen’s library.  Again, this technique is platform specific, so each has a different way to achieve this goal.

Ion Torrent “Template Preparation by Emulsion PCR” or “Clonal Amplification”

In the Ion Torrent method of template preparation, the multiple copies are created on an Ion Sphere Particle or ISP.  This looks like a bead with primers all over the surface of it.  Eventually this ISP will be deposited in a well on a chip and be sequenced.  In order for this ISP to create enough of a signal to be detected by the instrument, it must have many copies of the fragment all over the surface of the ISP.

At the beginning of the clonal amplification step, a specific concentration of combined libraries is added to the instrument, along with all the components of a standard PCR (buffer, dNTPs, polymerase) with the addition of the Ion Sphere Particles, which provide the primer, and oil.  The primers on the ISP are complementary to one of the adapters added during library preparation so that only the universal primer is necessary on the ISPs, instead of each individual gene-specific primer.  Through a series of steps, ideally, what is produced is a droplet of oil containing one ISP, one sample’s amplicon, and the components of the master mix.  This, along with millions of other ISPs in droplets of oil, will undergo cycles of PCR, with the primers on the ISP priming the specimen’s amplicon.  These amplicons will replicate all over the ISP, and as a final step, NaOH will be added to separate the strands.  The strands that are not anchored to the ISP by the universal primer will be lost, leaving each ISP single stranded and ready for priming in the sequencing step.



One thing to consider is the concentration of the combined libraries that are added at the beginning of the template preparation.  If the concentration is too low, obviously not enough amplicons will be amplified on the ISPs, and the end result will be not enough data.  Conversely, if the concentration is too high, there is a possibility of more than one sample amplicon ending up in the droplet of oil.  In the end, more than one fragment gets amplified on the ISP.  This ISP is called “polyclonal” and the data from it will get thrown out.  Optimizing the concentration takes a few runs and the concentration can be different for each instrument in the lab.

Illumina MiSeq “Cluster Generation by Bridge Amplification”

Illumina’s method of template preparation is termed cluster generation by bridge amplification and actually takes place on the MiSeq a step before the sequencing step.  The multiple copies are created in close proximity to each other, just as with clonal amplification, but instead of using a separate ISP for each specimen, a separate location on the flow cell is used.  A flow cell is essentially a glass slide that has universal primer anchored all over it.   This universal primers are, again, complimentary to the adapters added during the library preparation.  The combined libraries are flowed over the slide at the beginning of the run and they anneal to the universal primer.  The fragment then folds over and anneals to the second universal primer.  This strand is then replicated.  After replication, the strands are denatured creating two single strands.  These then replicate again, thus producing a cluster of the same fragment in a localized area on the slide.  This occurs for each specimen’s amplicons all over the slide.  At the end of the cluster generation step, the reverses are all cleaved off leaving only the single stranded forwards ready for sequencing.

Top View of Flow Cell After Cluster Generation – Each color represents one amplicon of one specimen

Concentration is just as important in this setup as in the Ion Torrent setup.  If the concentration is too high with this assay, the clusters generated will be too close together on the flow cell, thus the sequencing signal from each cluster will overlap.  The data generated from these areas will not be able to be discerned so it will get thrown out.

Join me next quarter for the next installment – sequencing!



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

Which Potassium Do You Believe?

A patient presented to the Emergency Department at the St. Paul’s Hospital. Initial blood was collected by phlebotomy staff (one poke) at 6:55 am in the morning and the specimen was received in the lab at 7:11 am.

Venous blood gases: Potassium  7.3 mmol/L
Plasma Lytes: Potassium  3.3 mmol/L

Emergency phoned the lab about these discrepant potassium results. What is going on?! The venous gas specimen was centrifuged and appeared hemolysed (3+), while the plasma sample had no evidence of hemolysis.

The phlebotomist indicated there was no problem with the collection. Repeat testing was initiated an hour later.

Venous blood gases: Potassium  6.4 mmol/L
Plasma Lytes: Potassium  3.6 mmol/L

The venous gas specimen was centrifuged and appeared hemolysed (3+).

Because the venous gas specimens were transported on ice and the other tubes of blood collected were sent at room temperature, the biochemist discussed the possibility of a red cell cold agglutinin with the ER physicians. The ER physicians requested evaluation for a cold agglutinin (the EDTA tube collected for early hematology was used for this analysis). Lab staff performed the screen and it was 4+ for cold agglutinin.  ER physicians were advised to believe the lower potassium results and to avoid sending further specimens on ice for this patient.



-Dr. Andrew Lyon, PhD, FCACB, DABCC is a clinical chemist and clinical toxicologist. He is the current past-president of the Canadian Society of Clinical Chemists. He is currently Division Head of Clinical Biochemistry of the Saskatoon Health Region and teaches general pathology residents as a clinical associate professor of Pathology and Laboratory Medicine at the University of Saskatchewan.