Transfusion Medicine Case Study: Positive Pretransfusion Test

A 72 year old man was admitted to the hospital for an aneurysm repair. The physician ordered a type and crossmatch for 6 units of blood in preparation for surgery. The patient history included surgery in 2016 during which he was transfused with 4 units of RBCs.

patient’s blood type: A positive

antibody screen: negative

history: anti Jkb  (2016)

6 Jkb negative units were found and full crossmatches were performed. One of the 6 donor units was incompatible. What is the most probable explanation for these findings?

If the patient has a negative antibody screen, and no history of an antibody, most facilities would do an electronic crossmatch or an immediate spin crossmatch. The immediate spin (abbreviated) crossmatch will simply verify ABO compatibility. However, if the patient has a positive antibody screen, we must identify the antibody, phenotype the patient, and do a full AHG crossmatch with donor units that are antigen negative for the corresponding antibody. In this case, the patient had a history of an antibody, so the antibody must be honored, and antigen negative units must be chosen for transfusion.

Kidd antibodies demonstrate dosage, are often weak, and can be found in combination with other antibodies. Because if this, they can be notoriously difficult to detect. They are usually IgG and are made in response to transfusion or pregnancy. Jkb has an antigen frequency of about 73% in the white population and about 43% in the black population. To find antigen negative blood, we consider that about 27% of units would be antigen negative. The tech working on the sample screened 21 units and found 6 that were Jkb negative.

AHG crossmatch results:

unit 1: compatible

unit 2: compatible

unit 3: compatible

unit 4: 3+ at AHG

unit 5: compatible

unit 6: compatible

There are 2 possible scenarios for the above results. A crossmatch is a test between donor’s red blood cells and patient’s plasma. Antigens, we know, are on red blood cells and antibodies are detected in the plasma. So, with a negative antibody screen, crossmatch incompatibility is due either to a patient antibody to a low incidence antigen on the donor red blood cells, or a donor cells with a positive direct antiglobulin test. We can easily rule in or out a positive donor DAT by performing a DAT on the segment. If the donor unit has a positive DAT, the unit should be quarantined and the positive DAT reported to the collecting facility. If the donor unit has a negative DAT, the patient likely has an antibody to a low incidence antigen.

Low frequency antigens are uncommon, but antibodies that recognize them are less rare. Fortunately, for patients with these antibodies to low frequency antigens, finding antigen negative compatible blood is easy. As we can see, 5 of the 6 chosen units were negative for the unknown low frequency antigen and were antiglobulin crossmatch compatible. The low prevalence of the antigen makes compatible blood readily available. If transfusion is necessary, it should not be delayed while waiting for identification of the antibody.

In this case, the antibody screen was repeated and the negative result was verified. In many cases, it may not be possible for a lab to identify the antibody because the lab may not have the necessary panel cells or typing reagents. Yet, these antibodies to low incidence antigens that react at AHG can be clinically significant and cause severe hemolytic transfusion reactions. To identify the antibody, you may need to send the sample to a reference lab for testing against a panel of reagent red cells that express low incidence antigens. Alternately, the donor red cells that were incompatible can be tested against known antibodies to low prevalence antigens  to help identify the antibody.

In this patient, anti-Wra was identified. The incompatible donor unit was verified to be Wra positive. Wra is part of the Diego system, usually IgG, and has ben implicated in hemolytic transfusion reactions.

One of the reasons I have written up this case is questions my Transfusion Medicine students often ask about exam and exam prep questions concerning incompatibility. Below are 2 questions to give examples of the confusion.

“At the indirect antiglobulin phase of testing, there is no agglutination between patient serum and screening cells. One of 3 donor units was incompatible.. The most probable explanation for these findings is that the:

  1. patient has an antibody directed against a high incidence antigen
  2. patient has an antibody directed against a low incidence antigen
  3. donor has an antibody directed against donor cells
  4. donor has a positive antibody screen”5

answer: b

“Which of the following would most likely be responsible for an incompatible antiglobulin crossmatch?

  1. recipient’s red cells possess a low incidence antigen
  2. anti-K antibody in donor serum
  3. recipient’s red cells are polyagglutinable
  4. donor red cells have a positive direct antiglobulin test”4

answer: d

I am asked why is one answer “low prevalence antigen” and one answer “positive DAT”? I typically ask questions of my students to let them reason out the answer. Take a careful look at the words antigen and antibody. Remember that a DAT is a test of red cells, the IAT tests for antibodies in plasma. A crossmatch uses donor red cells against patient plasma. Therefore, even though these are both reasons for the incompatibility of one out of multiple units, each question only has one answer of a common reason for such incompatibility. Be sure to read questions and use your theory and knowledge of testing when encountering discrepancies and problems in Blood Bank. To all of my students: Happy Studying for your ASCP exam!

References

  1. Fung, Mark K., Technical Manual 18th ed, Bethesda: AABB, 2014.
  2. Harmening, Denise M. Modern Blood Banking and Transfusion Practices, 7th edition, 2019.
  3. Schonewille, Henk, et al. “The importance of antibodies against low‐incidence RBC antigens in complete and abbreviated cross‐matching”. The Journal of AABB. 20 June 2003.
  4. BOC Study Guide, 5th edition. Clinical Laboratory Certification Examinations.  ASCP, 2016
  5. BOC Study Guide, 6th edition. Clinical Laboratory Certification Examinations.  ASCP, 2018

-Becky Socha, MS, MLS(ASCP)CM BB CM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 30 years. She’s worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

2020 Vision

Welcome back everybody! Thank you for all the engagement on my last post, Up In Smoke¸ where I discussed the plenary publications surrounding the vaping crisis and EVALI as new pulmonary pathology entity. This month, let’s start 2020 off right. After the holiday break and going on some of my last pathology residency interviews, I’d like to reflect on this new year by taking a look at 20 exciting things on the horizon for those of us in pathology and laboratory medicine!

So, let’s take a look at 2020 with some 20/20 vision… (sorry, not sorry)

20. Big, big, big, big data

Image 1. Can’t mention databases without this invaluable website that has made me look somewhat semi-competent in many instances! Thanks Pathology Outlines (Source: um, pathologyoutlines.com)

Last year, Elsevier’s Clinical Solutions Director in China discussed three topics that would impact our profession in 2019—so let’s start there. These first three go hand-in-hand in prepping the stage for 2020. Up first: the never-ending explosion of biomedical information and the continuing tidal wave of health data we don’t even know what to do with just yet! It’s a very interesting estimate that, by 2020 (aka now!) the whole sum of medical knowledge will double every 73 days. How on earth are we to manage, when compared to 1950 it would have taken 50 years to double? Well, the argument in the linked Elsevier blogpost discusses how evidence-based inquiry databases will store and organize this knowledge for us: think UpToDate, or ExpertPath, or ImmunoQuery…some of you are nodding your heads in relief, great, I’ll move on.

19. Precision Medicine

Image 2. PD-L1, or programmed death ligand 1, is one of several new targets for cancer therapy that utilized cellular checkpoints in cell cycles alongside T cell and NK cell functional immunity to fight cancer a little more precisely than classic chemotherapy regimens. Look at you, all up to date, and stuff. (Source: AstraZeneca graphic, azimmuno-oncology.com, content)

The second topic last year’s Elsevier’s blogpost discussed was the growth and rapid development of highly specific, targeted, individualized treatment plans. The mainstay example is of course how oncology treatments are moving away from one-size-fits-all chemotherapies to individualized mutation-specific immunomodulating therapy. (We’re moving like melting glaciers but moving nonetheless.) I was definitely well equipped with my ASCP online CE credits as I found myself discussing testing patients during my heme/onc training for PDL-1 and other tailored targets. We’re just starting to ride this wave and it’s definitely growing fast.

18. AI in healthcare (part 1)

Image 3. Artificial Intelligence is getting really good at pattern recognition. Why did I choose this picture? Oh, because it’s a study of how China-based JF Healthcare, a Siemens off shoot start up AI group, designed an algorithm that beat radiologist at Stanford on precision, delivery, and accuracy. Woah. (Source: hitconsultant.net)

Yep. I went there—it’s exciting! But notice I’ll come around after some other topics to really get into the heart of AI in path. Basically, the last point in the blogpost discussed the way smart software has been growing in medicine; particularly with radiology and surgery, using advancements in robotics and detection software to predict and stratify clinical information for patient care. Within this context let me quote them directly for you, “…there remains some uncertainty around the role of AI and its true impact on pathology, it is important to recognize that AI-based technologies or machines will never replace pathologists. Instead, such innovations will play an assistive role, augmenting the decision-making capabilities of pathologists and helping them perform better and faster…” All my pathologist friends may now exhale. It’s going to be okay. We’ll talk more about this at #10.

17. New Tech, New Toys

Image 4. You don’t have to go far to read about new tools and new tech. How’s this: a saliva-based rapid Malaria detection assay, courtesy of our friends at ThePathologist.com. New, rapid, accurate and deliverable diagnostics…now within spitting distance. Nailed it.

Pathologists are like the 007’s of the clinical team…at least when it comes to developing tech. There are so many new gadgets and tools we clinicians have available to us today. I delivered a recent TEDx talk where I discussed the “unrecognizable future” of medicine—and obviously now look for new and exciting ways to tell people I gave a TEDx talk. The important thing is that 73 days of doubling medical knowledge is happening so fast we don’t even know what we have available to us! Finger-print drug tests, smartphone facial capillary blood pressures, liquid biopsies, virtual MS-based immunohistochemical stains that never actually stain a single cell, cytology AI, deep data mining of free text pathology reports…it’s not a short list. It’s exciting, and we should all be sharing and collaborating to use these exciting tools together in creative ways for positive outcomes!

16. No More Silos

Image 5. Business and management have long discussed the importance of tearing down silos of knowledge in order to improve workflow and outcomes. It’s a growing conversation in healthcare and we’ve got our own isolated pockets of data that need to come out to the forefront, too. (Source: ERP Consulting, Estes Group Image, estesgrp.com)

Last year, I wrote a few pieces here on Lablogatory that mentioned High-Reliability Organizations (HROs) which require absolutely full sharing of responsibility as well as knowledge in order to solve problems and improve patient-care outcomes. There are many ways siloed thinking can harm the progress of any institution. It takes leadership, creative problem-solving, transparency, and teamwork. In the coming months of 2020, keep an eye out for pockets within your organizational environment that act as black holes or veils to keep pieces of critical data from the rest of the team. Encourage discussions between you and your peers, check biases about what you think might be important for one team vs. another, and try to share successes and failures as a group.

15. New Types of Colleagues

Image 6. We’re all different. And that’s ok. Each one of us is a brain, and an athlete, and a basket-case, a princess, and a criminal. Okay maybe not the last one, but we can all contribute in some important meaningful way. (Source: The Breakfast Club, 1985)

What I just mentioned about engaging in new conversations with folks you might not have worked with before—its not groundbreaking, its just good practice! In order to tear down #16’s silos, we’ve got to seek out and explore new ways to collaborate with colleagues outside our everyday scope. There will always be discussions about generational divides and differences that create culture strife in the workplace, or political/opinionated schisms that divide even the most cohesive of medical specialties. (I’m looking at you ACOG, ACP, ASCCP, and others: it’s Cervical Cancer Awareness Month, can we just agree on some guidelines already…) Soapbox over. But seriously, this isn’t a new concept. Feel like a lab half filled with boomers and millennials can’t make the cut? Well, the Harvard Business Review gave us great recommendations for this exact type of interpersonal growth exercise—in the NINETIES! The take home message: having an open culture and proactive leadership allows for fruitful exchange and growth!

14. Digital Pathology!

Image 7. Bigger, I want these screens bigger! The desk of tomorrow’s anatomic pathologist might have less glass and more pixels, screens, and queued data with high-output servers that are stocked with smart software to sift out normal results so they can focus on really tough morphologies. Maybe even with augmented reality software, or other crazy stuff I can’t think of yet! (Source: Inspirata, digital pathology)

It’s coming. You can’t stop it. It’s exciting. I don’t care what you think. Well I actually do care, but don’t knock digi-path till it grows into whatever it’s going to become. The desk of the (anatomic) pathologist-of-the-future will look a lot different from today and that’s really cool. Once upon a time, a very long, long time ago—in the eighties maybe—radiologists still had films where we actually used radiation to change the exposure of images to be read over a light box. Classic scene, right? Doctor, the x-ray is ready! *THWIP* *CLICK* *BUZZ* and cue the contemplative stare on the wall light. Then, they went digital and get to hang out in the dark with four computer screens and coffee, and really comfy chairs. I mean what a form of progress, can’t deny.

13. MS Methodologies

Image 8. Okay, MSI crash-course time. All you really need to know is that this method allows for great specimen preservation on tiny samples, high resolution, the ability to combine with molecular testing, and fascinating implications for margin detection, mutation analyses, and more! (Source: https://blog.waters.com/molecular-visualization-ms-imaging-delivers-insights-for-cancer-research)

In my mailbox this month, is another excellent edition of The Pathologist and in it there’s a great article on Mass Spec imaging transitioning from a research tool to a clinical one. Woah. We’ve all talked about and praised MALDI-Tof methods for microbiologic assays and detection, but the expanse of mass spectrometry has developed rather quickly. Now, it’s looking for a niche in routine laboratory diagnostics outside of the old chemistry analyzer… It’s a new, non-destructive way of examining tissue and gleaning data from the smallest pieces of gross specimens. We’re onto something here, keep an eye on MSI.

12. Molecular—Need I say More?

Image 9. Move over International Space Station, the folks at Thermo Fischer Scientific want to share their take on the Next Generation of molecular testing. (Source: The Pathologist)

Same edition of The Pathologist, about 25 pages back: a discussion on the value of molecular Next Generation Sequencing. I’ve already bored half of you, wait! Come back. I agree with you, you can only call it Next-Gen so many times before a whole generation of laboratorians get bored of talking about new tumor markers or mutations. But what’s happening with NGS testing that you should know? Simply put, there are NGS analyzers that are faster, with smaller footprints, combined with smarter software that is making molecular more feasible for laboratories that used to shy away from the notion of including NGS or LDTs in their lab testing menus. This means more labs, running more molecular, for more specific populations, in real time that can collaborate with that many more new colleagues while breaking silos—well just look up at #18, 17, 16, and 15!

11. Global Health

Image 10. From Dr. Razzano’s post on Lablogatory

Dr. Dana Razzano recently interviewed me for her global health series, and we got the chance to talk about the important intersection of laboratory medicine and global public health. Getting involved in a community—especially for those of us in healthcare—often includes a survey of what kind of health challenges you face. For some it’s access to clean resources like water, for others it’s a complex system of reimbursement and billing issues that complicate delivery of care, or even more basic assessments reveal high rates of local infections with preventable illness. But you can’t tackle infrastructure change, political reform, or vaccine education single-handedly. Global health is an increasing part of our global world and, if we stay true to our professional values, we should be at the forefront.

10. AI in Healthcare (part 2)

Image 11. Drawing to represent AI from my TEDx talk, Unrecognizable Medicine 2019, TEDxAUCMED

Oh I told you I’d come back to this. Some folks are still apprehensive about AI—that’s ok—I am too, but only because I want to make sure it’s done right. Don’t expect any Skynet stuff, we’re not going that deep. So let me tell you some of the things I got to see on the residency interview trail that piqued my interests. At one hospital system, I saw plans for their anatomic pathology department to go fully digital with augmented AI software to help score mitoses and other morphologic traits by 2025. At another institution, I saw plans for data mining historical free text pathology reports to predict and stratify future specimens before they even got signed out! At a third system, I saw the utilization of smart software to predict clinical lab values for a patient’s personalized reference range…pre-analytically! This stuff is coming in hot so watch for it! What AI-related advancements are you seeing in your neck of the lab?

9. Patient Consultation

Image 12. Courtesy of SUNY Upstate Pathology Department via Twitter, a newly renovated pathology residency review room and patient consultation suite for the dedicated purpose of this invaluable interaction.

Another thing noteworthy of my residency trail are institutions which are championing the face-to-face consultative role of the clinical pathologist in patient care. We, at the end of the day, are consultants to all; physicians and patients alike. And many in our field are celebrating this role by pushing the envelope toward a progressive and effective future for pathology and laboratory medicine at large.

8. Graphic Medicine

Image 13. My very first #pathdoodle – What do Pathologists do?

Graphic medicine is fantastic! I wish I could have discovered this years ago. Nevertheless, in the last two years I have sought out and read numerous pieces which bridge a significant gap between clinician and patient. And if we in laboratory medicine plan to reach patients more directly, we shouldn’t be afraid to come up with creative solutions to communicate with them. My (current) approach, #PathDoodles which I post occasionally on social media, attempts to explain concepts in pathology to the everyday lay person. What will you do to reach your patients?

7. Wellness Programs

Image 14. Anonymous survey responses to Dr. Saint Martin’s program from trainees at Loyola University Medical Center. (Source: The Pathologist)

Another interesting thing I encountered on the residency interview trail was the way in which different programs addressed the concept of wellness. Some did, some did not, but everyone discussed it. Because, after all, it is important; not just for residents and physicians, but medical lab scientists, support staff, and …yes… even administrators. Work and work-life stresses and balance take a psychological and emotional toll, and in order to be happy and healthy we need ways to strengthen our mental health along the way. Last year, Dr. Marissa Saint Martin won a 2018 award from the American Association for Physician Leadership for her work in preparing residency resilience and promoting wellness through structured curricula at Loyola Health System and Mount Sinai in Miami. She’s been featured with ASCP numerous times as well as in The Pathologist. It’s promising to see such strong support for residency trainees; keep an eye out for other praise-worthy programs this year!

6. Social Media

Image 15. It takes a new kind of clinician to serve a new kind of patient. How will you rise to meet the challenges on the horizon that we can’t predict yet?

You caught me. I can’t make any predictions about the future of health care delivery, education, or collaboration without touting the importance of social media. As a member of the official ASCP Social Media Team (Go #ASCPSoMeTeam!), I’m a proud contributor not only to the content that we publish online, but to the message that unifies and spreads our #StrongerTogether mantra. Social media is fantastic tool to reach new colleagues, spread new ideas, and make new advancements in both our field and in reaching people where they are.

5. Moving Out of the Basement

Image 16. Is this closer to a representative image of your lab than you’d wish? Don’t worry, you’re not alone. Basement labs are so last season. (Source: Seret Studios, Bridge Basement, Brooklyn NYC)

One of the most memorable interviews I had this season was with a transfusion specialist who recounted to me his memory of training in pathology during his early days of residency. Green and eager, he told me how he remembered being taken on a tour of the facilities to be shown his desk/work space and upon reaching an underground level of the hospital building a sign read ‘BASEMENT’ with an adjacent one ‘PATHOLOGY’ …he paused to say it out loud, “What’s a basement-pathologist?” We bonded over the stifled stereotypes in our work and shared stories of great and terrible lab spaces we’ve seen. Some programs are renovating, some (for better) are moving on up… How are your facilities?

4. The Pipeline Problem

Image 17. The …scope of a pathologist has changed over time (see what I did there?). Sure, lots of us push glass and diagnose entities on tissue samples with complex investigations and work up, but many more of us ensure quality laboratory efficacy, develop new cutting-edge tests, manage donor centers and transfusion protocol, address infectious or public health crises, and more! I think the more we celebrate our profession, the more will join and contribute to our #StrongerTogether culture!

Well I mentioned stereotypes. I’ve talked about it before.  You already know about shortages in pathology from physicians to lab scientists. And don’t get me started on pay—especially for lab workers and those in forensic path! But this is a topic I think we’re going to see a boon in media coverage in the coming years, starting now. Some of our awesome colleagues are talking about the “pipeline problem” from a myriad of angles: addressing medical student clerkships, exposure to laboratory workflow in school curriculum, advertising the infinite possibilities of careers within our profession…and more. CAP, ASCP, USCAP, and other professional societies have done amazing work in their due diligence to represent our field and advocate for the advancement of our work and image. A former CAP president once said, “Pathology is what a pathologist does,” and well, it’s a good start. Where do you see solutions to our pipeline problem?

3. PA’s and… CPA’s?

Image 18. Pathologist assistants work directly under the supervision of a pathologist in a similar way that physician assistants work with physicians. Clinical pathologist assistants help to determine the cause of disease through the examination of blood and other bodily fluids and tissues. (Source: UAB)

Now this, this is a cool concept and it’s the first time I’ve seen it. We all know and love our pathology assistants (PAs)—especially resident pathologists—as they make the training, education, and workload a better process for learning. They have awesome training and operate essentially as highly specialized clinicians in anatomic pathology. But what about the clinical, laboratory medicine side of things…? I recently saw a program advertised at the University of Alabama at Birmingham that trains clinical PAs for consultative and ancillary support roles to the clinical pathologist! Fantastic! Read more about it here! There are a growing number of DCLS (doctor of clinical laboratory science) professionals, now we’ve got a new pipeline to invite talented folks to work with us in a new way.

2. I’ll Have a Residency!

Image 19. Need I say more? What does the paper say?? Where am I going to end up? It’s a whirlwind of a season and a crazy system—once in a lifetime sort of adventure! Stay tuned, I’m sure I’ll have a post or ten about the next chapter of my career… (Image source: AAMC)

I have seen so many fantastic programs across the US these past few months, and so many decisions go into the way medical graduates rank programs to match into. To learn how the AAMC/NRMP matching system works, watch this video (maybe with some calming tea). We’ll see where I end up matching to, but I’m excited for the next chapter and to really dive into pathology even more!

1. You!

Obviously, the most important thing to keep an eye out for this year is you! Your goals, your ambition, your plans, your ideas and thoughts, and all the ways you contribute to our fantastic profession! I encourage you all to share, collaborate, and be creative with the ways in which we advance the future of laboratory medicine and continue to keep in mind that we do these things for our patients everywhere.

Thanks for reading, see you next time!

Constantine E. Kanakis MD, MSc, MLS (ASCP)CM completed his BS at Loyola University Chicago and his MS at Rush University. He writes about experiences through medical school through the lens of a medical lab scientist with interests in hematopathology, molecular, bioethics, transfusion medicine, and graphic medicine. He is currently a 2020 AP/CP Residency Applicant and actively involved in public health and education, advocating for visibility and advancement of pathology and lab medicine. Follow him on Twitter @CEKanakisMD

Biomarker Testing for Cancer Patients: Barriers and Solutions, Part 2

As you may recall last month I shared common barriers to biomarker testing for cancer patients in the community. I also began to dive-in to a few solutions that I have seen implemented to overcome the barriers. Last month I shared solutions that may help with high cost and long turnaround times for biomarker testing. This month I would like to discuss issues with tissue including quantity.

Here are the top 10 barriers that I’ve seen to biomarker testing in the community:

  1. High cost of testing.
  2. Long turnaround time for results.
  3. Limited tissue quantity.
  4. Preanalytical issues with tissue.
  5. Low biomarker testing rates.
  6. Lack of standardization in biomarker testing.
  7. Siloed disciplines.
  8. Low reimbursement.
  9. Lengthy complex reports.
  10. Lack of education on guidelines.

Sample quantity and quality are both important when considering biomarker testing. If we don’t have enough material we cannot perform the test (quantity not sufficient or QNS). If we have poor quality we cannot trust the results. The old adage of garbage in garbage out holds true for biomarker testing just as it does for all other lab tests.  

I’ll start with sample quantity this month and cover quality issues next month. The issue here is that a variety of biopsy types are performed on patients depending on the location and size of a suspicious mass. Historically we only needed enough material for the pathologist to make a diagnosis. Now we often need enough material for diagnosis and biomarker testing. Some tumor types such as breast and ovarian cancers produce enough material in locations that are easily accessible that tissue quantity is rarely an issue, however other tumor types such as lung and pancreatic cancers there is often an issue with tissue quantity. These tumor types must be handled with care to ensure no tissue recovered is lost.

The first step in addressing tissue insufficiency is knowing where you are starting. Do you have an issue with quantity not sufficient (QNS) rate? If you don’t know how many of your cases are insufficient for biomarker testing, then you can’t determine if you have an issue. If your testing is performed at a reference laboratory, you can request your QNS rate from the lab. They may also be able to provide you with the national QNS rate and then you could benchmark yourself against your peers. It is important to have an accurate QNS rate, so if there are blocks that are not sent to the reference lab because the pathologist has determined the block to be exhausted (no tissue is left) then the QNS rate provided by the reference lab may be artificially low.

It is important to agree upon what is QNS. We consider a specimen to be QNS if we cannot perform biomarker testing on the block. Others may consider the block QNS only if there wasn’t sufficient material for diagnosis. We have to ensure there is enough tumor content in the tissue to proceed with biomarker testing, in our case 10% of the nucleated cells (not volume) must be tumor (determined by pathology review of an H&E slide). If we have enough tumor, we can still end up with a QNS block due to low DNA and RNA yield. So we need sufficient tumor and sufficient tissue. 

Here is a brief overview of solutions I have seen work to address limited tissue that can lead to high QNS rates:

  • Education. The person collecting the biopsy needs to understand how much material is needed. Remember we have moved the goal post. Sufficient material for diagnosis was enough in the past, now we need more material to perform biomarker testing. Educating the team on why we need more material is valuable in ensuring sufficient material is collected.
  • ROSE. Rapid onsite evaluation (ROSE) by a pathologist in the procedure room to determine sufficiency has been shown to decrease the repeat biopsy rate [1]. The pathologist can ensure the biopsy is being collected in a tumor rich region and help ensure areas of necrosis are avoided.  
  • Embedding cores separately. We often get core needle biopsies on lung cancer specimens. We prefer 3-5 cores. It is best practice to independently embed the cores in separate blocks. I have also seen labs that embed no more than 2 cores in one block. This would allow one block to be conserved for diagnosis and the other to be used for biomarker testing.
  • Visual cue for limited tissue. Someone far more creative than me developed a process in histology where in cases of limited tissue the tissue was embedded in a red cassette. This cassette color was a visual cue for everyone handling the block that the tissue was limited and care should be taken when facing into the block. This has evolved over time to a red bead being embedded beside the tissue. Any visual cue and an associated procedure to ensure tissue conservation can help ensure we are conserving tissue in cases where it matters.
  • Limited IHC Stains. The primary reason a biopsy is performed is for diagnosis. It is recommended that as few IHC stains as possible be used to make the diagnosis. This will conserve tissue for biomarker testing.
  • Unstained Slides. Cutting 15-20 unstained slides is considered best practices in tumor types such as lung where biomarker testing will be performed within 30 days. Long term storage of unstained slides is not recommended.
  • Reduce the number of times the block goes on the microtome, because every time the block is put back on the microtome it must be refaced. This results in wasted tissue. This can be prevented by thinking ahead and cutting everything you know will be needed while the block is on the microtome.

References

  1. Collins BT, Murad FM, Wang JF, Bernadt CT. Rapid on-site evaluation for endoscopic ultrasound-guided fine-needle biopsy of the pancreas decreases the incidence of repeat biopsy procedures. Cancer Cytopathol. 2013;121:518-24.

-Tabetha Sundin, PhD, HCLD (ABB), MB (ASCP)CM,  has over 10 years of laboratory experience in clinical molecular diagnostics including oncology, genetics, and infectious diseases. She is the Scientific Director of Molecular Diagnostics and Serology at Sentara Healthcare. Dr. Sundin holds appointments as Adjunct Associate Professor at Old Dominion University and Assistant Professor at Eastern Virginia Medical School and is involved with numerous efforts to support the molecular diagnostics field. 

Microbiology Case Study: A 60 Year Old Male Status-Post Orthotopic Liver Transplant with Headache and Word-Finding Difficulty

Case History

A 60 year old male with a past medical history of ulcerative colitis requiring total proctocolectomy and immunomodulatory therapy followed by an anti-Tumor Necrosis Factor α blocker for the last two years and primary sclerosing cholangitis with subsequent decompensated cirrhosis that ultimately required an orthotopic liver transplant on tacrolimus and prednisone for immunosuppression presents 17 days post-transplant with worsening headache for two weeks with associated word finding difficulty and expressive aphasia.

Laboratory and Diagnostic Findings

Brain magnetic resonance imaging demonstrated, a “Heterogeneous, partially hemorrhagic and centrally necrotic mass within the posterior left temporal lobe…infectious etiologies such as pyogenic/non-pyogenic abscesses to include fungal organisms, are highest on the differential” (Image 1). At the time of admission, his complete blood count demonstrated a leukocytosis (16.48×109 cells/L), anemia (hemoglobin of 7.8 g/dL, hematocrit of 24.8%) and a normal platelet count (367×109 cells/L). The automated differential showed 82% neutrophils, 10% lymphocytes, 6% monocytes, 1% eosinophils, and 1% basophils. A lumbar puncture was performed to obtain cerebral spinal fluid (CSF) and the analysis showed a glucose of 60 mg/dL, protein of 34 mg/dL, nucleated cell count of <1, and 6 red blood cells (completely normal CSF indices). Broad spectrum antimicrobials (Vancomycin, Piperacillin/Tazobactam, Metronidazole and Micafungin) were initiated. A 1,3-β-D-glucan test had a result of >500 pg/mL in both serum and CSF. Galactomannan, Histoplasma urine antigen, Cryptococcus antigen and other fungal testing were negative. Antifungal therapy was changed to voriconazole. Craniotomy was determined to be the best course of action and the patient was taken to surgery for debridement and pathologic evaluation.

Frozen section evaluation during the time of surgery showed granulomatous inflammation. Septate hyphae were observed on the fungal smear. Following surgery, amphotericin was added. Histologic evaluation of the tissue submitted from surgery showed pyogranulomatous inflammation with pigmented, spore-like structures present in multinucleated giant cells on hematoxylin and eosin (H&E) stain (Image 2). Grocott’s methenamine silver (GMS) stain also highlighted short segments of septate hyphae (Image 3).

Cultures from the surgical debridement grew a mould with central pigmentation (Image 4). Direct microscopic examination of the mould revealed thick-walled, oblong conidia with 3-5 cells, and uniformly pigmented hyphae (Image 5). A germ tube test showed germ tubes originating from both ends of the conidia consistent with Bipolaris species.

Image 1. T1-weighted (left) and T2-weighted (right) magnetic resonance imaging of the brain demonstrating a left temporal lobe mass.
Image 2. Hematoxylin and Eosin stained photomicrographs showing pyogranulomatous inflammation with giant cell formation and circular structures within them (left) (40x objective magnification). The right shows gold-brown pigmented structures within granulomatous inflammation (40x objective magnification).
Image 3. Grocott’s methenamine silver stain highlighting short segments of irregular septate hyphae in the brain debridement specimen (10x objective magnification).
Image 4. Mature wooly brown-black colony on potato dextrose agar.
Image 5. Photomicrograph of a lactophenol blue tape prep of the mature fungal colony. Pigmented hyphae and short 3-4 cell conidia are readily identified (40x objective magnification). This specimen also tested germ tube positive (not shown), indicating that this dematiaceous fungus is Bipolaris spp.

The patient’s mental status significantly improved following surgical debridement, 2 weeks of liposomal Amphotericin B, as well as long term treatment with voriconazole. The voriconazole was later switched to posaconazole due to concerns for fluoride toxicity. He completed a year of posaconazole with significant improvement of the abscess observed on imaging and resolution of headaches with no other visual problems. He continued to recover cognitive function with some residual difficulty with reading, comprehension and speech that eventually resolved.

Discussion

Phaeohyphomycosis refers to infections caused by dematiaceous fungi that exist in a variety of forms when seen in tissues and commonly involves skin, soft tissue and nasal sinuses.1 In rare cases, central nervous system (CNS) involvement has been reported. CNS phaeohyphomycosis is predominantly seen in immunosuppressed patients; however, cases involving immunocompetent individuals do exist.2 In one case series from Houston, Texas, five of seven cases of cerebral mycosis were caused by a dematiaceous mould.3 Interestingly, the patient presented in this case came to medical attention around the Dallas-Fort Worth area of Texas.

Cladophialophora bantiana is the most common dematiaceous fungus associated with CNS phaeohyphomycosis, but rare cases of Bipolaris species have been reported previously in literature.4-6

We report a case of CNS phaeohyphomycosis by Bipolaris species following orthotopic liver transplant with an excellent patient outcome. This case is unusual, in part, because the typical hospital course of a patient with phaeohyphomycosis is generally dismal.7 The stories of successful treatment often involve complete debridement of discrete lesions.7-8 In our case, the patient underwent surgical debridement and treatment initially with liposomal Amphotericin B and later transitioned to long term therapy with newer azole antifungals.

References

  1. Revankar SG, Sutton DA, & Rinaldi MG, (2004). Primary Central Nervous System Phaeohyphomycosis: A Review of 101 cases. CID, 38, 206-2016
  2. Filizzola MJ, Martinez F, & Rauf SJ, (2003). Phaeohyphomycosis of the central nervous system in immunocompetent hosts: report of a case and review of the literature. Int J Infec Dis, 7, 282-286
  3. Raparia K, Powell SZ, Cernoch P, Takei H, (2010). Cerebral mycosis: 7-year retrospective series in a tertiary center. Neuropathology, Jun; 30(3): 218-223.
  4. Frank T, Esquenazi Y, Nigo M, Wanger A, Portnoy B, & Shepard S, (2016). Disseminated Phaeohyphomycosis with Brain Abscess and Biliary Invasion Due to Bipolaris spp. In an Immunocompetent Patient. Annals of Clinical & Laboratory Science, 46(4). 
  5. McGinnis MR, Campbell G, Gourley WK, & Lucia HL, (1992). Phaeohyphomycosis Caused by Bipolaris spicifera, An Informative Case. Eur. J. Epidemiol, 8(3), 383-386
  6. Rosow L, Jiang JX, Deuel T, Lechpammer M, Zamani AA, Milner DA, Folkerth R, Marty FM, & Kesari S, (2011). Cerebral phaeohyphomycosis caused by Bipolaris spicifera after heart transplantation. Transpl Infect Dis, 13, 419-423.
  7. Dixon DM, Walsh TJ, Merz WG, McGinnis MR, (1989). Infections due to Xylohypha bantiana (Cladosporium trichoides). Rev Infect Dis, 11: 515-525.
  8. Gadgil N, Kupfermen M, Smitherman S, Fuller GN, Rao G, (2013). Curvularia brain abscess. J Clin Neurosci, Jan;20(1): 172-175.

-John Markantonis, DO is a second year Clinical Pathology resident at UT Southwestern in Dallas. He has interests in Medical Microbiology and Transfusion Medicine.

-Dominick Cavuoti, DO is a Professor at UT Southwestern in the Department of Pathology. He is multifaceted and splits his time as the Medical Director of the Parkland Hospital Clinical Microbiology Laboratory and Parkland Cytology attending among other administrative and educational activities.

-Clare McCormick-Baw, MD, PhD is an Assistant Professor of Clinical Microbiology at UT Southwestern in Dallas, Texas. She has a passion for teaching about laboratory medicine in general and the best uses of the microbiology lab in particular.

A New MLS Graduate's Experience

I wrote last as a student in the medical technologist program at NorthShore University Healthcare System in Evanston. Now, as my first post as a certified medical technologist, I wanted to share what the journey was like becoming certified, finding a job, and transitioning into the professional arena.

Throughout the program, I felt relatively confident in the material and what we were learning and applying in rotations. We took about 1000 tests over the course of the program – at least that’s what it felt like. When the ASCP BOC exam began peaking its head, I wasn’t too intimidated. To me, it was just another test.

The first step was registering for the exam. My intention was to take the exam the day after I graduated, but I was bad and waited one month prior to graduation to register (it can take up to 45 business days to process). Don’t do as I did! Thankfully, the process was quicker than expected and I was only delayed one week after graduation.

The last four weeks of my program were intimidating to say the least! We had cumulative finals in addition to simulated board exams. Therefore, my BOC exam studying began through preparing for these. One of the most valuable resources that I cannot recommend enough is LabCE by MediaLab. I first discovered LabCE through their manual UA and differential simulators, but then discovered that they have testing simulators which our program director used to create practice exams for subjects and for the BOC exam. Their questions range in difficulty and each one has an explanation, which presents a great way to study (at least for me). It helped me develop study guides on material I consistently got wrong or completely forgot.

Two weeks before my exam, I discovered the BOC CLS study guide. I immediately paid for overnight shipping and received it the next day. This turned into my main study tool – I do best quizzing myself, then reading up on topics I got wrong. I would be lying if I said I felt confident when I received that book and went through the first 50 questions. I felt incompetent. Despite my previous review and studying, I felt as if I discovered an entirely new language. The book is very detailed and covers everything from a to z for laboratory science, with some topics only being covered briefly in school. As time went on, the shock factor wore off and I continued to focus on the things I no longer remembered and believed were important.

Despite the endless hours studying, I felt that there was much left to cover and the night before my exam I remember feeling overwhelmed. There is only so much information the human brain can store without the hands-on experience that ingrains what you learn. Throughout the test I felt as if I were failing, something that seems to be common place among BOC test takers. When it was time to see my score, my hands became clammy. Despite the suspense, I passed! When I received my scores later, I did much better than expected!

So, my concluding advice while preparing for the BOC exam is to focus on summarizations of your notes and to review all your formulas throughout your program. Go through as many practice exams as possible to help you see where you’re lacking and to prepare you for the wording on the BOC exam. Throughout my program, I would type up one to two-page notes for exams for later review. These were helpful when I had to go back and review things I did not remember. Additionally, despite the amount of studying you do, there is bound to be information you will not know and that’s okay. You know more than you think you know and through review you will only increase the recoverable information that is already in your head.

Lastly, I wanted to speak about the process of a new grad finding a job and transitioning from a student to a health professional. As I went through rotations, my passion for each specialty changed. At first, I wanted to be a generalist because I wanted to be more marketable and do everything. Then it was blood bank, then micro, and then came molecular (yay!). I began my job search about 2 months before graduation, and applied for those jobs about a month and a half before graduation. Being a soon-to-be new grad, I knew that I might not end up in the exact field I desired right away (which was molecular). I applied for mostly blood bank, micro, and molecular jobs – as these were of the most interest to me.

I applied for about 6-7 jobs in total and I ended up discovering, and eventually obtaining, my current position as an HLA molecular scientist at Northwestern’s transplant lab in Chicago. Throughout the interview process, being 100% honest of what you do and don’t know is the most important advice. Most employers ask a lot of detailed questions only to gauge where they need to start in your training. A good rule of thumb to remember is that if you’re a new grad being interviewed, then the employer is already okay with the fact that you don’t have much experience or knowledge of the specialty.

Before starting, a lot of people warned me about specializing immediately after graduating. While I hear their concerns, for me I plan on staying in the molecular field for the rest of my career – there are many opportunities and molecular is only becoming more and more advanced/widespread.

Now that I am 2 months into my job, I have fallen in love with it. There is endless opportunity to continue learning and to challenge myself. Walking into this specialty, I had two HLA lectures and nothing more. While my first month and a half mostly consisted of DNA isolation and cell lineage DNA isolation for chimerism tests, I have finally started training on an assay and data analysis for engraftment monitoring (chimerism). As a new grad in such a specific specialty, I have accepted that there will be a large learning curve. My advice is to keep your mind open to learning new things and fuel your motivation to learn more and more. Never stop asking questions and never turn down resources others hand you that have helped them.

As I gain more experience in the HLA world, I plan on writing articles tailored to this field and sharing what I learn. I hope my experience as a new grad helps others approaching this new time in their lives and gives them a sense of direction/confidence.

-Ben Dahlstrom is a recent graduate of the NorthShore University HealthSystem MLS program. He currently works as a molecular technologist for Northwestern University in their transplant lab, performing HLA typing on bone marrow and solid organ transplants. His interests include microbiology, molecular, immunology, and blood bank.

Microbiology Case Study: 18 Year Old Male with Shortness of Breath

Case History

A 18 year old male with no significant past medical history presented with 2-3 month cough, hemoptysis, fevers, night sweats and 15 pound unintentional weight loss. He originally started to feel mild shortness of breath with activity following his return from the Pacific Northwest. He was diagnosed with pneumonia and given antibiotics. He denied vaping, hookah use or any smoking. Of note, his maternal grandmother had pulmonary tuberculosis two years ago, but at that time he had a negative interferon-gamma release assay (IGRA). Since that time he had traveled extensively including Europe and Asia.

Upon admission, physical examination showed decreased breath sounds and coarse crackles in the right lower lung field. His blood test results were unremarkable. Chest X-ray and CT scan showed diffuse reticular pattern and a 4 cm cavitary lesion in his right lower lobe (Image 1). Brochoalveolar lavage fluid, sputum as well as blood were submitted to microbiology lab for bacterial, fungal and mycobacterial culture.

Image1. Chest X-ray and CT showed diffuse reticular pattern with a 4cm cavitary lesion in his right lower lobe.

Laboratory results and management

The patient’s Quanti FERON®-TB Gold test (an IGRA) was positive.Laboratory studies including fungal culture to look for endemic fungi and HIV were negative. Auramine-rhodamine (acid fast) staining of sputum smear showed 4+ acid fast bacilli (AFB). Mycobacterium tuberculosis was confirmed by both our laboratory developed TB-PCR assay and a commercial TB-PCR assay (Xpert MTB/RIF). DNA sequencing was performed by the Centers for Disease Control and Prevention (CDC) in order to predict antibiotic susceptibility profiles for first-line anti-tuberculosis drugs including Ethambutol, Isoniazid, Pyrazinamide, and Rifampin. Because the result showed drug-susceptible tuberculosis, the patient started the first-line anti-tuberculosis drugs. Four days after admission, the patient discharged home with follow up with the department of health. After six weeks incubation, the sputum culture grew Mycobacterium tuberculosis with a pan-susceptible antibiotic profile.

Image 2. Ziehl-Neelsen stain of acid fast bacilli (AFB) on sputum smear. Image from the CDC website.

Discussion

Mycobacterium tuberculosis (MTB) remains a global health problem. The continuing spread of drug-resistant tuberculosis is one of the most difficult challenges for MTB control. The CDC recommends rapid laboratory confirmation of MTB with using a nucleic acid amplification test (NAAT), followed early drug susceptibility testing. Our case is consistent with the previous reports that NAAT can identify MTB and DNA sequencing can determine the drug susceptibility within a few days. Our case can be summarized into two points.

First, early NAAT provided diagnosis MTB within 2 days after patient admission, enabling the timely initialization of infection control measures. Although culture remains the gold standard for laboratory confirmation of active TB infection, it can take 6-8 weeks to grow in a culture media since MTB is slow growing pathogen. Early laboratory confirmation of MTB with NAAT led rapid initiation of the patient treatment and transmission interruption.

Second, DNA sequencing guided us to start an optimal anti-tuberculosis treatment within 3 days because of the accurate prediction of susceptibility profiles for first-line anti-tuberculosis drugs. In comparison, the conventional susceptibility testing with the drug-containing medium requires 1 month to complete beyond initial growth and identification of the organism. Recent studies demonstrated high accuracy of genotypic predictions (>91.3 % sensitivity; >93.6% specificity). Our case supports a theory that DNA sequencing can help to determine which anti-tuberculosis drugs should be used for treatment.

Despite the notable advantage, NAAT has limitations. There is a relatively low positive predictive value in smear-negative pulmonary MTB. Recent meta-analysis found that overall sensitivity of NAAT to be 90.4% for diagnosis of pulmonary MTB. The sensitivity of the NAAT further defined to be lower in smear negative (75%) compared to smear positive (98%) pulmonary MTB. Additionally, cost effectiveness of NAAT and DNA sequencing is another concern. Further assessments of the benefit of NAAT and DNA sequencing utilization for smear negative MTB are needed.

In conclusion, our case demonstrated that NAAT and DNA sequencing was beneficial to reduce the time to initiation of an optimal MTB management.

References

  1. Prediction of Susceptibility to First-Line Tuberculosis Drugs by DNA Sequencing. N Engl J Med 2018; 379: 1403-15.
  2. Use of Nucleic Acid Amplification Tests in Tuberculosis Patients in California, 2010-2013. Open Forum Infect Dis. 2016 Oct; 3(4): ofw230.
  3. Assessment by Meta-Analysis of PCR for Diagnosis of Smear-Negative Pulmonary Tuberculosis. J Clin Microbiol. 2003 Jul; 41(7): 3233-3240.

-Sachie Ikegami MD, PhD is a 1st year anatomic and clinical pathology resident at University of Chicago (NorthShore). Sachie’s academic interests include neuropathology and molecular pathology. She is passionate about understanding how pathology informatics improving clinical practice. Outside of the lab, she enjoys jogging.

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois. Follow Dr. McElvania on twitter @E-McElvania.