Hematology Case Study: The Story of the Platelet Clump: EDTA-Induced Thrombocytopenia

I belong to a Hematology Interest Group and always enjoy seeing the case studies and questions that other techs post. This group is multinational so I see posts from techs all over the world. It’s interesting to see the similarities and differences in standard operating practices and the roles techs play in different areas and different countries. It’s also interesting to see that we all come across the same types of problems and difficult specimens! In the last few months in this Hematology Interest Group, I have seen many questions and comments about resolving clumped platelets, and am therefore using this opportunity to shed some light on these tricky specimens. The case I am presenting, and the photos, are courtesy of Abu Jad Caesar, who is a Lab manager at Medicare Laboratories – Tulkarm branch, in Palestine.

The patient had a CBC performed on a Nihon Kohden 6410. WBC was 12.7 x 103μL, impedance platelet count was 20,000/μL on initial run, other parameters appeared within normal limits. The sample was warmed and a Na Citrate tube was requested to rule out pseudothrombocytopenia. After warming, the EDTA was rerun with a platelet count of 0/μL. The Na Citrate tube was run, and platelet count from the instrument was 189,000/μL. (Figure 1) Because of the blood:anticoagulant ratio in the Na Citrate tube, a multiplier of 1.1 was applied, thus making the Na Citrate platelet count 207,900/μL. Slides were made, stained and examined. Image 1 shows the clumping in the EDTA tube. Image 2 shows the smear from the Na Citrate tube, with no visual clumping.

The CBC was reported with the following comments: Platelet clumping observed, 2 samples drawn to rule out thrombocytopenia. EDTA whole blood smear had many platelet clumps noted (EDTA induced thrombocytopenia). Conclusion: Platelets are adequate and estimated to be about 200,000/μL.

Figure 1. Results from warmed EDTA tube (left) and Na Citrate tube (right).
Image 1. Clumped platelets seen with EDTA.
Image 2. Normal platelet count with no clumping seen with Na Citrate.

Platelet counts in the normal range don’t usually give us too much trouble in reporting, even if some clumping is present, mainly because they are normal. Adequate platelet counts fall within a typical reference range of about 150- 450 x 103/μL. If there are instrument flags for a platelet abnormal scattergram or platelet clumps, it is recommended to repeat testing by another method. If the initial count is performed by impedance counting, many analyzers can also report optical or fluorescent platelet counts. With impedance counting, very small RBCs or fragments may be counted as platelets, thus giving a falsely increased platelet count. With optical counting, large platelets can be counted as RBCs, thus giving a falsely decreased count. Some Sysmex hematology analyzers use impedance and optical counts and also feature fluorescent platelet counts which use a platelet specific dye and give accurate platelet counts without the interferences of other methods. A normal platelet count, even with clumping seen on a smear, is still usually estimated to be normal (or may occasionally be increased.)

Thrombocytopenia, on the other hand, can be a challenge in the hematology laboratory. With thrombocytopenia, physicians need an accurate count to diagnose, treat or monitor patients. Even a small increase or decrease can be significant when there is a severe thrombocytopenia. With fewer platelets, every platelet counts!

One of the first questions we must ask with an apparent thrombocytopenia is if this is a true thrombocytopenia or if it is pseudothrombocytopenia (PTCP). A true thrombocytopenia represents a patient with a low platelet count who may need monitoring or medical intervention. It can be dangerous to miss true thrombocytopenia but is also dangerous to report a low platelet count in a patient with a spurious thrombocytopenia who is not actually thrombocytopenic. Pseudothrombocytopenia, or spurious thrombocytopenia, is defined as an artificially or erroneously low platelet count. In PTCP, the low platelet count is due to clumps that are counted as 1 platelet. (These large clumps can also be counted as WBCs, thus giving a falsely increased WBC count.)

We can divide PTCP into 2 categories Platelet clumping is most commonly caused by pre-analytic errors such as over-filled or under-filled EDTA tubes, clotted specimens, or a time delay between sample collection and testing. Techs should check the tube for clots and sample volume and do a delta check to help differentiate thrombocytopenia and PTCP. But, with an apparent ‘good’ sample, the next step would be a smear review. If there are clumps seen on the smear, then we need to decide what caused the clumps. Is it the first category, one of these common pre-analytical issues, or is it the 2nd category of PTCP, an in vitro agglutination of platelets? Conditions that can cause this in vitro agglutination of platelets include cold agglutinins, multiple myeloma, infections, anticardiolipin antibodies, high immunoglobulin levels, abciximab therapy and EDTA induced pseudothrombocytopenia. (EDTA-PTCP) Of these, EDTA induced pseudothrombocytopenia is the most common cause. (Nakashima, 2016).

When techs talk about platelet clump issues, it is usually because we are looking for ways to resolve or to accurately estimate the platelet count in these samples, and there doesn’t seem to be one easy answer. The clumping makes precise counting impossible and even estimates can be very tricky. How can we estimate these counts? Do we simply report the presence of clumping with “appear normal”, “decreased” or “increased”? Or, should we break our estimates into more ranges to give physicians more valuable information? And, what if the provider wants an actual count in order to give the patient the best care possible and we can’t resolve the clumping? What can we do to provide a count? Some of the first steps recommended include vortexing the sample for 2 minutes to break up platelet clumps, then re-analyzing. Warming samples may also help to resolve platelet clumps, particularly in samples with cold agglutinins or that have had a delay in testing and have been transported or stored at room temperature or below. If clumps persist and recollecting the sample still yields platelet clumping, then pre-analytical error can be ruled out an EDTA induced pseudothrombocytopenia may be suspected. Many labs will have an alternate tube drawn or use another method to help resolve the clumping.

So, what is EDTA induced thrombocytopenia (EDTA-PTCP)? This is not representative of a particular clinical picture, and is not diagnostic for any disorder or drug therapy, but is a laboratory phenomenon due to presence of EDTA dependent IgM/IgG autoantibodies. These antibodies bind to platelet membrane glycoproteins in presence of EDTA. EDTA induces and enhances this binding by exposing these glycoproteins to the antibodies. (Geok Chin Tan, 2016) Though it is an in vitro phenomenon, patients with certain conditions, such as malignant neoplasms, chronic liver disease, infection, pregnancy, and autoimmune diseases, do have increased risk of EDTA-PTCP. However, EDTA-PTCP has also been observed in patients who are disease free. (Zhang, 2018)

What are some alternate methods to help resolve EDTA induced platelet clumping challenges? Probably the most common is to redraw the sample in a Na Citrate tube. Both EDTA and Na Citrate tubes should be drawn. In a true EDTA-PTCP, as seen in our case study, you should see clumps on the smear made from the EDTA tube and no clumps on the smear made from the Na Citrate tube. Because of the volume of the anticoagulant in the Na Citrate tube you must also apply the dilution factor of 1.1 to the count from the Na Citrate tube to get an accurate platelet count. Note, however, that hematology analyzers are FDA approved and validated for use with EDTA tubes. If you wish to use a different anticoagulant, the method must be validated in your laboratory. Note also that alternate methods will generally only resolve EDTA -PTCP, and not clumping due to other cold agglutinins, medication or disorders. In addition, anticoagulant induced thrombocytopenia is not limited to EDTA. It can also occur with citrate and heparin. In a study, it was found that up to 17% of patients with an EDTA -PTCP also exhibited this phenomenon with citrate. In fact, researchers have found, and we have found in our own validations, that some samples that do not clump in EDTA actually DO clump in Na Citrate. Thus, alternate tubes may not resolve all platelet clumping. (Geok Chin Tan, 2016)

Some labs have validated ACD (Citric acid, trisodium citrate, dextrose) anticoagulant tubes for EDTA-PTCP. Using this method, the EDTA tube and ACD must be run in parallel and a conversion factor applied, reflecting the difference in sample dilution in the 2 tubes. A parameter such as the RBC must be chosen to make this comparison. Using a formula that divides the RBC in EDTA by the RBC in ACD gives a ratio that reflects the dilutional differences between anticoagulants. This ratio can then be multiplied by the ACD platelet count to obtain the ACD corrected platelet count. (CAP Today, 2014). Some sources have recommended ACD tubes because the incidence of clumping with Na Citrate can be frustratingly high. It is theorized that the more acidic ACD tube may prevent platelet clumping better than Na Citrate. (Manthorpe, 1981)

Less commonly used tubes are CTAD (trisodium citrate, theophylline, adenosine, dipyridamole) and heparin. CTAD acts directly on platelets and inhibits platelet factor 4 thus minimizing platelet activation. Downsides to CTAD tubes are that they are light sensitive and must be stored in the dark, and can be costly. They also alter the blood/additive dilution ratio so calculations must be used, as seen with Na Citrate and ACD. Heparin tubes are less commonly found to be beneficial in resolving platelet clumping issues because heparin can active platelets. Heparin tubes are also more expensive, so have not generally been a first choice for EDTA-PTCP.

I have heard from techs that their labs have very good results using amikacin added to EDTA tubes to prevent spuriously low platelet counts in patients with EDTA-PTCP. Amikacin should be added to the EDTA tube within 1 hour after draw and testing is stable for up to 4 hours at room temperature. Results of a study done in 2011 showed that the addition of amikacin to the EDTA tube produced rapid dissociation of the platelet clumps with little or no effect on morphology or indicies. This method has proved very promising for reporting accurate platelet counts in patients with multianticoagulant induced PTCP. (Zhou, 2011)

The last anticoagulant tube that I have seen mentioned by many techs in the hematology interest group are Sarstedt ThromboExact tubes. I have seen many posts from techs who use these and they seem to have a very good success rate. ThromboExact tubes contain magnesium salts and are specifically designed to determine platelet counts in cases of PTCP. They are currently validated only for platelet counts and samples are stable for 12 hours after collection. Interestingly, before automated hematology analyzers, magnesium was the anticoagulant of choice for manual platelet counts. EDTA-PTCP has been recognized since EDTA automated platelet counts were introduce in the 1970s. A 2013 study in Germany used ThromboExact tubes with excellent results for resolving multianticoagulant induced PTCP. These tubes became commercially available during the study, in 2013. (Schuff-Werner, 2013) Unfortunately for us in the United States, these tubes are not available in the US. I was recently at a conference and went up to the Sarstedt representatives and asked about these tubes. I was told that they are available in parts of Europe and Asia but are not FDA approved in the US. I asked very hopefully if they were looking at getting FDA approval and was unfortunately told that “they didn’t think they had the market for them to pursue approval.”

Whichever alternative method your lab chooses to use, it is recommended to draw an EDTA and the alternate tube together. This way the 2 counts and the presence or absence of clumping in the tubes can be compared. We have many patients who had one incidence of clumping, yet when the provider orders a Na Citrate platelet count, we get a new draw of both EDTA and Na Citrate tubes together, and there is no flagging or clumping seen with EDTA. In these cases it is appropriate to result the EDTA results as there is no evidence of EDTA-PTCP.

When a patient has a low PLT count without any hematologic disease, family history, and/or bleeding-tendency identified, and pre-analytical errors have been ruled out, PTCP should be considered. This does not mean that a patient with PTCP will have a normal platelet count after the clumping is resolved. As stated above, many patients with EDTA-PTCP have hematological or other disorders and may be truly thrombocytopenic. Resolving the clumping in these patients allows us to give the provider an accurate platelet count, which is very important in thrombocytopenic patients.

The flow chart below (Figure 4) shows some things to consider when dealing with platelet clumping. It is our goal to resolve clumping so that we can report an accurate platelet count in a timely fashion. In the laboratory where I work, I have validated Na citrate tubes, but these seem to resolve clumping in less than 50% of patients. As a last resort, to get an accurate platelet count, some articles have suggested collecting a fingerstick and performing manual counts. I did include this in the chart as an option for multianticoagulant PTCP, however, due to the difficulty in collecting a good specimen and the subjectivity of counts, along with problems associated with necessary calculations, our pathologists have decided that we will not do manual platelet counts. For this reason, I am currently involved in platelet clumping monitoring and will be conducting a small internal study to compare ACD, CTAD and Na Citrate tubes in parallel. Depending on those results we may also then test amikacin. If we come to any enlightened conclusions I’ll write another short blog with our results!

Thanks again to Abu Jad Caesar, lab manager at Medicare Laboratories – Tulkarm branch, in Palestine, who provided me with this textbook perfect case of PCTP, which was easily resolved by collecting in Na Citrate. We wish they all read the textbooks and were as cooperative!

Figure 2. Flowchart for resolving and reporting of thrombocytopenia.

References

  1. CAP Today, January 2014. accessed online http://www.captodayonline/qa-column-0114
  2. Manthorpe R, Kofod B, et al. Pseudothrombocytopenia, In vitro studies on the underlying mechanisms. Scand J Haematol 1981; 26:385-92
  3. Nakashima MO, Kottke-Marchant K. Platelet Testing: In: Kottke-Marhchant K, ed. An Algorithmic Approach to Hemostasis Testing, 2nd ed. CAP Press; 2016:101
  4. Schuff-Werner,Peter, et al. Effective estimation of correct platelet counts in pseudothrombocytopenia using an alternative anticoagulant based on magnesium salt. Brit J of Haematol Vol 162, Issue 5. June 29, 2013
  5. Tan, Geok Chin et al. Pseudothrombocytopenia due to platelet clumping: A Case Report and Brief Review of the Literature. Case Reports in Hematology. Volume 2016
  6. Lixia Zhang, MMed,* Jian Xu, MD,* Li Gao, MMed, Shiyang Pan, MD, PhD. Spurious Thrombocytopenia in Automated Platelet Count. Laboratory Medicine 49:2:130-133. 2018
  7. Zhou,Xiamian, et al. Amikacin can be added to blood to reduce the fall in platelet count. Am Journal of Clinical pathology, Vol 136, Issue 4, Oct 2011.

-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.

A Med Tech Gives a TEDx Talk

Hello again everyone!

After a lot of positive responses and sharing on social media, my article last month got lots of people talking about annual meetings and how great they are for networking, learning, and advancing our profession. Not too long after the ASCP Annual Meeting in Phoenix, I was back in my Manhattan apartment working on my speech and graphics for a real life TEDx session hosted at my medical school.

Let’s pause here: if you either haven’t heard of the TED/TEDx brand or if you binge watch their 18 minute videos and want more links to watch now, now, now!

TED is a non-profit organization whose mission is to share “ideas worth spreading.” They’re about 35 years old and based in NYC stateside, and Vancouver in Canada. Basically, over the last few decades they hold conferences at those flagship sites called “TED talks” where selected speakers present on a myriad of topics. TEDx conferences are officially licensed but off-site events which operate under TED protocol and guidelines. There have even been spin-off conferences like TED MED, which focus solely on healthcare.

Image 1. What’s a TEDx talk? Basically, an off-site, officially sanctioned, “idea sharing” conference.

Some of the students at AUC School of Medicine, organized such a conference with official TED licensing and recruited me to join their list of speakers to deliver talks on their chosen theme: resilience. Officially called TEDxAUCMed, this conference included community members, students, artists, activists, and more discussing the human capacity for resilience in ways not commonly discussed. “Weathering the Storm” was the official event title, as the school located in the island nation of St. Maarten displays daily resilience especially since being hit by Hurricane Irma in 2016. Among their list of incredible speakers, I was humbled to be included! I titled my talk “Unrecognizable Medicine” and wanted to deliver a talk to students, clinicians, and those of us in medicine witnessing first-hand a tidal wave of new technologies and paradigms that redefine the way we discuss health. Oh, and since I’m a huge fan of #GraphicMedicine more and more each day, I hit that hashtag hard and decided to illustrate my whole talk!

Image 2. Title Card from my TEDx talk.

So what did I talk about, exactly…and what’s the big deal? I’m not going to re-hash my presentation for you in text—that’d be boring, and I’m obviously going to put a link at the bottom for you to watch it yourself. I got you, lab fam! But essentially, what I set up was a three-tiered template to assess and navigate that tidal wave of tech. Tools, skills, and strengths—three things inherent to the practice of medicine in any specialty.

Image 3. Red back-ligting. So intense. Thanks for coming to my TEDx Talk, literally!

There are untapped topics in medicine which are looming over the horizon. As medicine continues to evolve and change, the problems we face and the needs we must meet will become moving targets. New specialties will emerge, and new technologies will replace centuries old tools we cling to today. A shift in thinking is both proactive and healthy in a profession that mandates our commitment to preserving health and quality of life. I have spent years battling stereotypes in medicine and hope to challenge the fabric that places individuals in professional or academic boxes. Fresh first-years at some schools are already using point-of-care ultrasounds (POCUSes) instead of stethoscopes—which student sounds like they have better info on morning rounds, a student who maybe kinda-sorta heard some non-descript murmur, or a mini-pocket echocardiogram with an ejection fraction of 45%? Stereotypes have too long shaped the way students choose specialties, equating some areas to colloquial high school cliques! No offense to orthopedics or dermatology. Troponins used to be something you could hang your white coat on, but not anymore. What do you do with a new 5th generation Trop of 39 with a delta of 18? ACS or acute MI? Cancer therapy is exploding with personalized treatments being added every day! Any student right now would impress their heme/onc attending on rounds if they suggested PDL-1 and other immunotherapy testing for patients with newly diagnosed lung cancers. *Deep breath*

Ok. My point is, tomorrow’s medicine is going to have a lot of different therapies, tools, and even vocabulary that schools may never catch up with. How do you prepare for this explosion of knowledge? You look to yourself to take an inventory of your strengths and use those to guide your clinical sails. Addressing stereotypes head-on, learning on the spot, dealing with complex identities in your patients, and always practicing with compassion will lend itself to staying ahead and staying fulfilled.

Image 4. If you’re drawing cartoons of pathologists for an educational series, you probably make them look like you. Or in this case me, I guess. Keep an eye out for my #PathDoodles on social media!

Pretty heavy stuff right? But there’s something else that caught my attention in reflection on the TEDx talk… I’ve searched the TED library of videos, and while there are plenty of doctors, scientists, and pioneers in research discussing medical ideas, I haven’t seen any medical laboratory scientists. If you find any, please correct me. But, as I understand it, it’s just me. And that’s something special.

Image 5. My wife and I check-in for rehearsal at the TEDxAUCMed conference in sunny St. Maarten.

There’s a culture shift in our profession, and a lot of us are talking about it. Pathology and laboratory medicine are stepping out from behind the healthcare curtain and asserting itself as a champion for patients, truth, and the importance of data-driven medicine. Not only do I talk to groups of folks every time I get a stage, but I use social media to reach clinicians and patients! Yes, I’m one of few medical students-turned-residency applicants who didn’t change their name to hide their online presence for the winter. But instead of a secret twitter hibernation, I’ve used social media as a tool to network, engage, and connect.

One of my favorite new projects is something I call #PathDoodles where I break down the aspects of pathology and some specialty topics for those outside of medicine (and sometimes just outside our profession). I’ve already covered things like “what is pathology?” and the importance of autopsies, the role of medical laboratory scientists, and I continue to add more regularly!

Image 6. One of a growing list of #PathDoodles.

There’s a culture shift in our profession, and a lot of us are talking about it. Pathology and laboratory medicine are stepping out from behind the healthcare curtain and asserting itself as a champion for patients, truth, and the importance of data-driven medicine. Not only do I talk to groups of folks every time I get a stage, but I use social media to reach clinicians and patients! Yes, I’m one of few medical students-turned-residency applicants who didn’t change their name to hide their online presence for the winter. But instead of a secret twitter hibernation, I’ve used social media as a tool to network, engage, and connect.

One of my favorite new projects is something I call #PathDoodles where I break down the aspects of pathology and some specialty topics for those outside of medicine (and sometimes just outside our profession). I’ve already covered things like “what is pathology?” and the importance of autopsies, the role of medical laboratory scientists, and I continue to add more regularly!

Follow me on Twitter (@CEKanakisMD) and check out my TEDx talk:

My talk begins at 5:00:00. Enjoy!

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

Global Health Narratives Interview Series: Meet Dr. Constantine E. Kanakis.

Constantine E. Kanakis, MSc, LS(ASCP)CM is a board certified Medical Laboratory Scientist and is a newly minted MD. Any pathology program director reading this should pay close attention, since he is currently completing the Pathology Match for July 2020 and you would be lucky to have him in your program. Constantine has done incredible work in the field of pathology already, and was recognized in the prestigious ASCP 40 Under Forty award program in 2017 and he was recognized as a Top Ten medical student with the ASCP Academic Achievement Award in the same year.It was his passion for working to better his community that earned him these, and I can only imagine what else he will do in his career. I came across his work through reading his ASCP Lablogatory blog contributions, to which he has contributed insightful and quality material for years. His work in global health particularly stands out as what he has been able to accomplish in such a short time and while in medical school is really spectacular. If you want to know how to get engaged in your community and make a tremendous impact in the world, read on, you will surely be inspired to do so after reading this!

Q: Can you tell me about your background and what led you into Pathology for your career choice?

A: I received my undergraduate and masters in Chicago studying molecular biology, political science, bioethics, and medical laboratory science. I’ve worked in various laboratory roles for the last ten years, mostly in blood bank and hematology. After some time, I decided to return to pursuing a more advanced career in medicine and go to medical school and was naturally drawn to pathology from having worked extensively in the lab.

Public health was always something I was interested in, but didn’t know how to get involved. This changed when I had an opportunity to take a service-learning elective course in medical school focused on community outreach. We were prompted to choose a project to focus on, and since Zika virus was such a heightened threat to the community of Sint Maarten in 2015-2016, as well as the region at-large, I decided to focus my efforts there. I organized an effort to reach out to the community and help educate them on Zika prevention/infection through speaking at town hall meetings, health drives, and by creating vector control projects in the field. Together with a team, we developed school-based task forces to educate children so they would bring the information home to their parents and siblings. This arm of the project was mirrored after the recycling initiative in the 90’s that was targeted at US school children to bring recycling programs into the home. Recycling started in schools and it was effective in changing the home culture. Our Zika education based program was so successful that the Sint Maarten Ministry of Health adopted it as an official outreach program as part of their Collective Prevention Services. And was even touted as a success by representatives at the 2016 Global Health Security Agenda session in Miami.

I also married this community outreach project to the Zika virus research that I was involved in with my medical school. We used commercially available antibody kits and I both wrote the SOPs and ran testing alongside other team members in the laboratory.

With my background in public health, research, and working in the laboratory as a technologist, Pathology is a career that will allow me to engage in all of these things. Pathology is a perfect career for focusing on global health due to its ability to intervene on behalf of the population in a data driven way. Rather than helping one person at a time, I can help entire demographics through epidemiologic based interventions. 

Constantine Kanakis (center right, first row) and the team in the fight against mosquito borne diseases.

Q: Why do you think medical students should get involved in global or public health?

A: Getting involved in solving the problems in your community enriches your education in a way that solely reading about issues cannot. When you are actively engaged in the solution, the problem becomes more than just something you are reading and learning about in the text. Not only does this enhance your education and understanding, but it also gives you the benefit of being part of your community in a meaningful way. There are so many preventable issues to focus on – in the US and abroad.

Q: How can someone get started in serving their community?

A: Start by looking around at your immediate surroundings and take an assessment of the issues affecting the community. Anyone can do this, whether you are a physician, scientist, or a community member. The first step is to collect data to define the issue and narrow your target. Next is to plan an intervention; start small and organize or join a group working on the issue and just get involved. You will be surprised at how quickly things can develop. And don’t be afraid of failure—taking setbacks are critical in a continuing process of reevaluating and readjusting your project!

Q: Now that you have finished with medical school, what is next for you and where do you see for your future?

A: In between residency program interviews this month, I will be flying to Sint Maarten to deliver a TEDx talk about the rapid evolution of medicine and how we can prepare for the changing landscape. [You can view the talk here: https://vimeo.com/365844585 (skip ahead to 5:00 to jump straight to Constantine’s presentation)].

Next I’m planning to present an abstract in the next Caribbean Center for Disaster Medicine conference. With hurricanes threatening the Caribbean islands and in particular Sint Maarten which was hit in 2016, there’s been a lot of energy centered around disaster preparedness. My focus is on making sure the planning efforts including blood bank and other lab services are ready in the case of a major disaster.

In the future, as a pathology resident and beyond, I want to continue to work in both my local US setting and abroad. In the US, there are many public health issues that need to be focused on. For example, there’s been a record resurgence in preventable infectious diseases due to the anti-vaccination movement. There are also people suffering from Hepatitis C related cirrhosis who aren’t aware that Hepatitis C is curable. There are many educational campaigns for issues like this that can change lives, and pathologists are the ones that can fulfill that role as health educators.

My wife is a RN, has a master’s of nursing science (MSN), is a certified nurse leader (CNL), and is finishing her Doctorate of nursing practice (DNP) in advanced public health with a focus on vulnerable populations and disaster planning, has been an excellent partner and resource for community outreach all along and we hope to focus on these issues throughout our careers. It’s exciting to think of all the possible ways we can help make our community better!

Constantine Kanakis delivering a recent TEDx talk.

-Dana Razzano, MD is a former Chief Resident in her fourth year in anatomic and clinical pathology at New York Medical College at Westchester Medical Center and will be starting her fellowship in Cytopathology at Yale University in 2020. She is passionate about global health and bringing pathology and laboratory medicine services to low and middle income countries. She was a top 5 honoree in ASCP’s Forty Under 40 in 2018 and was named to The Pathologist’s Power List of 2018 and 2019. Follow Dr. Razzano on twitter @Dr_DR_Cells.

Hemoglobin Electorphoresis in Children

This last month, I rotated through our Children’s hospital, which included reviewing hemoglobin electrophoresis tests. I’d learned about them before in residency, but they can be quite more interesting (complicated) than I expected.

Hemoglobin electrophoresis is a blood test to look at different types of hemoglobin to determine if there are any abnormalities. In a children’s hospital it is frequently ordered as a reflex for an abnormal newborn screen or when a child is incidentally found to be anemic. The test is performed in 2 stages. 1st lysed blood samples are run on gel electrophoresis and different types of hemoglobin are separated as they move at different speeds. Several types of hemoglobin will run within the same region, so a secondary method of separation is always employed.

Below, you can see how some bands in the same area of an acidic gel (agarose) are actually very different on the alkaline gel (cellulose acetate) and vice versa.

At our hospital, we use HPLC and measure retention times of the hemolysate to quantify and identify different hemoglobin types present. As a basic primer you should recall that hemoglobin is a tetramer with a pair of alpha globin + a pair of either beta, delta or gamma globin (each separate genes).

Alternative hemoglobins are enriched in populations where malaria is endemic as these variants may provide improved fitness by promoting resistance to the malarial parasite that reproduces inside red blood cells. Thus, many people of African or south east Asian descent may carry these variants.

Our case is that of a 2 year old girl with anemia who had testing sent by her primary care doctor for the following CBC:

This is indicative of microcytic anemia, but unlike some Thalessemias the RBC isn’t very high. More on this later.

Looking at the gel result, there is a large band in the area coinciding with Hgb C. We also see the normal Hgb A2 and a small amount of Hgb F. We know Hgb F can be increased in Hgb SS and thus could also be present if she had Hgb C trait or disease.

InkedBlog 1B_LI

Looking at the next HPLC result, we see there is a similar very high level of Hgb C (68%) with corresponding levels of Hgb F and Hgb A2 (note: acetylated Hgb F and Hgb F are added together). Thus, this fits with a homozygous C with some compensatory A1 and F, right?

Remember Hgb C is a β -globin variant and you only have 2 β -globin genes, so if you are homozygous for the C variant on the β-globin gene (HBB), then Hgb A1, which is made of normal β-globin would be impossible to produce. Also you might be bothered by all of these small peaks. However, there are often small peaks that can’t be definitively identified and are likely post-translationally modified hemoglobin. But in the context of an abnormal Hgb A1 that shouldn’t be there, we dug deeper.

One of the most common hemoglobinopathies is Beta Thalassemia (β-Thal), which clinically manifests when less of the beta hemoglobin protein is produced. Heterozygous mutations lead to Beta Thalassemia minor with minimal symptoms, while homozygous mutations lead to β-thal major with symptoms of anemia. Mutations in the β -globin gene, HBB, can lead to complete loss of β-globin (β0 variant) or partial of β-globin (β+ variant).

As this patient has less than 50% of Hgb A present (expected amount), they could also have a β+ variant as well. This would make them compound heterozygous for C and β+.

One of the hallmarks of Thalassemia is an increase in Hgb A2 (normal 2.5-3.5%). Hemoglobin A2 is a normal variant of A that is composed of two alpha and two delta chains (δ2α2). We see in our case that the Hgb A2 is normal at 2.5%. So it seems the patient doesn’t display a typical Thalassemia picture.

One condition that could create this scenario is if there is a variant in the delta chain of A2 that causes it to elute differently. Indeed, there is a delta variant that creates hemoglobin A2 prime (A2’) that moves near the S region of the HPLC. And when we look back at our unknown hemoglobins, Hgb X is marked at 1.03 of the S region and has an abundance of 3.9%. This supports it being the Hgb A2’ and if we add this together with the Hgb A2 we get an elevated 6.6% A2 total, which would be consistent with Beta Thalassemia. Lastly, one would wonder if we could find this third hemoglobin variant A2’ on the alkaline gel. Previous studies have shown the A2’ variant is more negatively charged, so on a basic gel, it should move further from the negative anode than the other hemoglobins. We don’t see anything to the left of the HgbC, but if we flip the gel over and look under the patient label, you can see a faint band that is likely the A2’!

In summary this case arose from 3 separate mutations in a single patient. She was compound heterozygous for a Hgb C and β+ variants in the β-globin gene and she was heterozygous for an A2’ variant on the delta-globin gene.  This was certainly a case where paying close attention mattered.

References:

  1. Abdel-Gadir D, Phelan L, and Bain BJ. Haemoglobin A2′ and its significance in beta thalassaemia diagnosis. Int J Lab Hematol. 2009 Jun;31(3):315-9. doi: 10.1111/j.1751-553X.2008.01038.x. Epub 2008 Feb 21.
  2. https://ghr.nlm.nih.gov/condition/beta-thalassemia

-Dr. Charles Timmons MD PhD is a pediatric pathologist at Children’s Medical Center in Dallas, TX. His responsibilities include signing out hemoglobin electrophoresis, HPLC and globin sequencing, and has been residency director for 17 years.

-Jeff SoRelle, MD is a Chief Resident of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and improving genetic variant interpretation.

Surgical Pathology Case Study: An Elderly Patient with Unexplained Pain, an Unremarkable CT Scan, and Enlarged Rugal Folds on EGD

Case History

The patient is a 72 year old woman who presented to her physician’s office with postprandial pain and unintentional weight loss. A CT scan was performed that showed no obvious abnormality or cause for the patient’s abdominal pain. The patient subsequently underwent an EGD and EUS which revealed enlarged gastric folds without hemorrhage. In addition, there was wall thickening seen in the body of the stomach within the luminal interface, superficial mucosa, deep mucosa and submucosa consistent with possible gastritis versus an infiltrative process. The remainder of the EGD and EUS was grossly unremarkable. These findings were concerning for possible linitis plastica. Pathology on the samples taken from the EGD were consistent with poorly differentiated adenocarcinoma that was invasive in both the gastric fundus and gastric body. The patient was initially taken to the operating room for a staging laparoscopy to ensure that there was no metastatic disease before beginning a preoperative chemotherapy regimen. The staging laparoscopy revealed a thickened gastric wall from the fundus to the antrum, consistent with linitis plastica, and no obvious evidence of metastatic disease. The patient then underwent peritoneal washings which showed no evidence of positive cytology. Based on these findings, the patient was started on a chemotherapy regimen of epirubicin, cisplatin and fluorouracil (5-FU), which she tolerated well. The patient was then taken to the operating room for a total gastrectomy procedure with Roux-en-Y esophagojejunostomy.

Diagnosis

Received fresh for intraoperative consultation is a total gastrectomy specimen with a black stitch designating the proximal side. It was requested by the surgical team to have the proximal esophageal margin frozen to ensure that esophageal tissue was indeed present, as well as to exclude the presence of any carcinoma. The proximal margin was negative for carcinoma with squamous mucosa present. The stomach measures 17.0 cm in length with an internal circumference ranging from 14.7 cm proximally to 9.0 cm distally. There is a 1.0 cm long portion of attached duodenum with an internal circumference of 5.8 cm. The serosal surface of the stomach is glistening, pink-tan and smooth with a scant amount of attached yellow, lobulated adipose tissue and omentum along the length of one entire edge measuring 26.0 x 13.0 x 1.0 cm. The stomach is opened to reveal glistening, tan mucosa with irregular rugal folds which are diffusely nodular, predominantly in the body of the stomach. There is a 6.5 x 5.0 cm are of flattened mucosa in the pyloric region (Image 1). The wall thickness measures 0.5 cm throughout. There are no grossly identifiable masses or nodules. Gross images are taken and the serosal surface is inked entirely in black. The adipose tissue is examined for candidate lymph nodes. Representative sections are submitted as follows:

B1 FS: Frozen section remnants

B2-B6:     multiple representative sections from the cardia

B7-B10:   multiple representative sections from the body

B11-B12:   multiple representative sections from the pylorus

B13:     representative perpendicular section through the distal resection margin

B14:     seven putative lymph nodes

B15:     five putative lymph nodes

B16:     three putative lymph nodes

B17:     seven putative lymph nodes

B18:     six putative lymph nodes

B19:     three putative lymph nodes

B20:     six putative lymph nodes

Histologically, the specimen consisted of diffuse, poorly differentiated, discohesive cells throughout all the layers of the stomach, penetrating into the serosa, with fibrosis, inflammation and signet ring cells present. In addition, angiolymphatic invasion was present. Based on the gross presentation and histologic appearance, the specimen was signed out as a diffuse gastric adenocarcinoma with a stage of T3.

Image 1.

Discussion

As of 2018, gastric cancer is the sixth most common cancer with approximately 1.03 million cases, and the third leading cause of cancer deaths worldwide, resulting in 783,000 deaths. Due to a better understanding of epidemiology, pathology, and molecular testing, as well as advances in new forms of treatments, the incidence and mortality in gastric cancer has been declining over the years. Of the gastric cancer types, rates of intestinal type carcinoma have been decreasing, however, the incidence of poorly cohesive gastric carcinoma (PCGC) and signet ring cell carcinoma (SRC) has increased. In order to accurately discuss PCGC, there must first be a discussion about the standardization of gastric cancer subtype definitions. Poorly cohesive, signet ring cell, and diffuse gastric carcinomas have commonly been used interchangeably. In 2010, the World Health Organization defined poorly cohesive gastric carcinoma as being composed of isolated or small groups of tumor cells. If there was a predominance of signet ring cells, then it would be termed a signet ring cell carcinoma. Mariette et al. proposed that a PCGC composed of 90% or more signet ring cells should be classified as SRC. The term “diffuse” corresponds to the same term “poorly cohesive”, and because of this, I will be using the term “poorly cohesive” solely going forward. In addition to this, the term “linitis plastica” would commonly be used interchangeably, but is best used as a term to describe the macroscopic appearance of PCGC or SRC.

Gastric carcinoma is classified as either early or advanced stage to help determine the appropriate type of intervention. Early gastric carcinoma is defined as invasive carcinoma confined to the mucosa and/or submucosa, regardless of lymph node metastases or tumor size. These tumors are generally smaller, measuring less than 5 cm in size, and found most commonly on the lesser curvature of the stomach at the angularis. Histologically, early gastric carcinoma will commonly present as well differentiated, mostly with tubular and papillary architecture. If the biopsies are composed of only mucosa, then distinguishing between well-differentiated carcinoma and carcinoma in situ or high grade dysplasia can be difficult. The presence of stromal desmoplasia in invasive carcinoma can help differentiate it from intramucosal invasion, which can contain single tumor cells within the lamina propria. This is an important distinction to make as intramucosal carcinoma does metastasize. Advanced gastric carcinomas will present grossly as either exophytic, ulcerated, or infiltrative tumors. Histologically, advanced gastric carcinomas will invade the muscularis propria and demonstrate cytologic and architectural heterogeneity, with a combination of patterns.

The 2010 World Health Organization classification determined four major histologic patterns of gastric cancer, which will often present with a combination of elements from the other patterns:

  1. Tubular: Most common pattern in early gastric carcinoma, with branching, distended or fused tubules containing intraluminal mucus, and nuclear and inflammatory debris
  2. Papillary: Most common in the proximal stomach with epithelial projections containing an underlying fibrovascular core. Also, it is frequently associated with liver metastases and an increased risk of lymph node involvement.
  3. Mucinous: Extracellular mucin makes up at least 50% of the tumor volume
  4. Poorly cohesive (including SRC): Mixture of signet ring and non-signet ring cells. Signet ring cells will have mucin pushing the nucleus to the periphery of the cell.

Helicobacter pylori (H. pylori) is a gram negative infectious bacteria that has been linked to gastric cancer. H. pylori is present in about half of the world’s population and other than gastric cancer, it is also associated with chronic gastritis, peptic ulcer disease, and gastric lymphomas. The bacteria is typically acquired during infancy and will remain for life if left untreated, with reactive oxygen species being generated that are capable of causing DNA damage due to the chronic infection. In addition, H. pylori can induce hypermethylation, resulting in the inactivation of tumor suppressor genes. Although H. pylori infection is considered a strong risk factor for developing gastric cancer, more commonly in intestinal type than diffuse type gastric cancer, only a small portion of those infected with the bacteria actually develop the malignancy. It is believed that approximately 80% of distal gastric cancers are due to a H. pylori infection, whereas there is little association between H. pylori and cardia gastric cancers.

In PCGC, such as this case, it is generally diagnosed in younger patients without a gender bias. Although PCGC can be associated with an H. pylori infection, it is more commonly related to a mutation in the tumor suppressor gene epithelial cadherin, also known as E-cadherin and CDH1. PCGC presents as an infiltrative growth of poorly differentiated, discohesive malignant cells that appear to arise from the middle layer of the mucosa. These cells can infiltrate as individual cells or as small clusters, but usually do not form glands (Image 2). If the gastric wall becomes extensively infiltrated by malignancy, the wall can be thickened and rigid, a macroscopic presentation termed as linitis plastica, which can lead to pyloric obstruction. Within PCGC, numerous signet ring cells can be present, leading to SRC. There is also a hereditary form of poorly cohesive gastric cancer referred to as hereditary diffuse gastric carcinoma, with an autosomal dominant pattern of inheritance. Histologically, it will include hyperchromatic nuclei, occasional mitoses, patchy intramucosal signet ring cells in the lamina propria, and carcinoma in situ associated with pagetoid spread of tumor cells along the preserved basement membrane. Hereditary diffuse gastric carcinoma will present with multifocal tumors under an intact mucosal surface, making diagnosis difficult. In patients with a CDH1 mutation and a family history of gastric carcinoma, a prophylactic gastrectomy is often the recommended treatment option.

Image 2.

References

  1. Adachi Y, Yasuda K, Inomata M, et al. Pathology and prognosis of gastric carcinoma well versus poorly differentiated type. Cancer. 2000;89(7)1218-24.
  2. Cancer. World Health Organization. Who.int. https://www.who.int/news-room/fact-sheets/detail/cancer. Published September 20, 2018. Accessed September 18, 2019.
  3. Carcas LP. Gastric cancer review. J Carcinog. 2014;13:14. Published 2014 Dec 19. doi:10.4103/1477-3163.146506
  4. Hu B, El Hajj N, Sittler S, Lammert N, Barnes R, Meloni-Ehrig A. Gastric cancer: Classification, histology and application of molecular pathology. J Gastrointest Oncol. 2012;3(3):251–261. doi:10.3978/j.issn.2078-6891.2012.021
  5. Mariette C, Carneiro F, Grabsch HI, et al. Consensus on the pathological definition and classification of poorly cohesive gastric carcinoma. Gastric Cancer. 2019;22(1):1-9 https://doi.org/10.1007/s10120-018-0868-0-
  6. Pernot S, Voron T, Perkins G, Lagorce-Pages C, Berger A, Taieb J. Signet-ring cell carcinoma of the stomach: Impact on prognosis and specific therapeutic challenge. World J Gastroenterol. 2015;21(40):11428–11438. doi:10.3748/wjg.v21.i40.11428
  7. Van Cutsem E, Sagaert X, Topal B, et al. Gastric Cancer. Lancet. 2016;388(10060):2654-64. https://doi.org/10.1016/S0140-6736(16)30354-3
  8. Weisenberg E. Diffuse (poorly cohesive) type carcinoma. Pathology Outlines. http://www.pathologyoutlines.com/topic/stomachdiffuse.html. Revised August 22, 2019. Accessed September 18, 2019

-Cory Nash is a board certified Pathologists’ Assistant, specializing in surgical and gross pathology. He currently works as a Pathologists’ Assistant at the University of Chicago Medical Center. His job involves the macroscopic examination, dissection and tissue submission of surgical specimens, ranging from biopsies to multi-organ resections. Cory has a special interest in head and neck pathology, as well as bone and soft tissue pathology. Cory can be followed on twitter at @iplaywithorgans.

An Introduction to Laboratory Regulations – Part III (Accreditation)

So far we have reviewed the different federal regulatory agencies responsible for establishing laboratory testing guidelines, a brief overview of the different roles each department plays, as well as a discussion on testing complexity. In today’s post we’ll cover the optional accreditations available to labs, and how accreditation differs from certification.

In the simplest of terms, certification is a mandatory requirement, whereas accreditation is optional. Certification is required in order for laboratories to receive payments from Medicare or Medicaid. Laboratories must meet the minimum requirements set forth by CLIA to earn and maintain their certification status.

Accreditation is an extra additional step that laboratories can take to set themselves apart from neighboring labs by holding themselves to a higher standard. Accredited laboratories must still adhere to the minimum CLIA requirements, but there are additional rules and requirements to be satisfied depending upon the different accreditation agencies.

More rules and paperwork, why would anyone volunteer to take that on? Depending on the size, complexity, and client population that your lab serves, the benefits to obtaining accreditation can greatly outweigh the challenges of maintaining that accreditation status.

One of the requirements to maintaining your CLIA certification is routine inspections to confirm compliance with the rules. Accreditation agencies require inspections as well, but thankfully in most cases your CLIA inspection can be satisfied by your accrediting agency; meaning your lab will receive a single inspection to satisfy both groups. Results will vary for each lab, but generally speaking the accreditation inspections are perceived to be easier to get through than those conducted by the federal inspectors. For example, agencies like The CAP and COLA tend to be more focused on sharing of ideas and good laboratory practices, rather than coming in as the “lab police” and looking only for problems. The explanation of their regulatory requirements tends to be more user friendly and easier to interpret as well, rather than the formal CLIA laws which are legal documents and read as such.

Recognition by an accrediting agency confirms that the laboratory is qualified and competent to perform testing for which it has received the accreditation for. This stamp of approval can help patients and clients feel comfortable in choosing your laboratory for their testing needs. For laboratories that perform testing as part of clinical trial evaluations, this can help reduce the number of requested on-site audits by the client themselves, as the client may choose to rely on the third-party accreditation assessment due to their high standards. It may also help encourage new clients to choose you for their testing needs, as the accreditation confirms your commitment to higher quality standards.

Another possible benefit of having accreditation status is the impact on your laboratory staff. Continually striving to raise the bar on your standards and going above the bare minimum instills a sense of professionalism in your employees. By continually reviewing the regulations and preparing for or responding to inspections, staff are more likely to be committed to complying with your organization’s quality management system and standards of performance. Staff who are familiar with the requirements and the reasoning behind why a certain task is performed or documented, are more likely to comply with those policies and procedures.

There are currently 7 CLIA approved accreditation agencies: https://www.cms.gov/Regulations-and-Guidance/Legislation/CLIA/Downloads/AOList.pdf. Some agencies are focused on a specific discipline, such as AABB for transfusion medicine, and others are more encompassing for all of the laboratory departments.  Organizations looking to become accredited should research each option in order to determine which ones would be best to meet their specific needs. It is also common for labs to maintain more than one accreditation at a time, for example AABB and CAP. As always, the regulatory agency with the most stringent rules would be the ones the lab is expected to adhere to. In cases of joint accreditation, multiple inspectors may be needed to complete the biennial inspection; however the agencies will try to coordinate efforts and work together so that the inspections occur simultaneously. Sticking with our AABB & CAP example, CAP will work with AABB to locate an AABB approved inspector for the transfusion medicine checklist, while the remainder of the CAP inspection will be carried out by CAP inspectors. The AABB inspector would then inspect the transfusion medicine department for compliance with both CAP and AABB requirements at the same time.

The accreditation process may be challenging, but once you have obtained that esteemed status, the opportunities for continual education and improvement of your laboratory will be endless.


-Kyle Nevins, MS, MLS(ASCP)CM is one of ASCP’s 2018 Top 5 in the 40 Under Forty recognition program. She has worked in the medical laboratory profession for over 18 years. In her current position, she transitions between performing laboratory audits across the entire Northwell Health System on Long Island, NY, consulting for at-risk laboratories outside of Northwell Health, bringing laboratories up to regulatory standards, and acting as supervisor and mentor in labs with management gaps.