Blood Splatter Does Matter

As a patient, when I go in to get my blood drawn, I have the anxieties and fears that most normally feel. Even as someone who understands the collection process and subsequent testing, it’s human nature to be nervous when having any type of medical procedure performed. Something most do not consider, however, is the fear and anxiety that the phlebotomist, nurse or laboratory technician who is drawing the blood may be feeling. Healthcare workers are at risk of biohazardous exposure daily and, in the wake of infectious diseases that can result in global pandemics, protection from this exposure is even more important. In the worst case, unanticipated patient movement or a combative patient may cause a needlestick injury to the person collecting. But what about the much less obvious risk of blood splatter? It’s important to understand all risks in order to put the appropriate protection in place. So, what exactly are the risks of blood splatter and how is the healthcare system working to minimize it?

Over the years, legislation such as the Needlestick Safety & Prevention Act and the Bloodborne Pathogen Standard has been put into place to decrease exposure risk, resulting in safety-engineered collection devices becoming mainstream. Prior to these changes, it was not uncommon for needles to carry no additional safety measures to protect healthcare workers and ensure the used needle was shielded when collection was completed. The rates of infection reflected this and, thankfully, have been significantly reduced with the introduction of safety devices.

While there have been several studies focused on needlestick injuries and the efficacy of safety engineered devices, there have been few on the potential for exposure through splatter when using these devices, though equally important. Splatter presents a real danger since it is known that infection can occur if mucous membranes are exposed to even minute amounts of blood. Most users may not even be aware that splatter or aerosolization has occurred and would not seek prophylaxis to prevent potential infection as a result. This would mean that they may not even be aware of a deadly infection taking hold in their body.

Though methods for assessing splatter may differ slightly, there have been studies that demonstrate visible and/or measurable splatter from use and activation of safety devices. One such study looked at retractable phlebotomy and intravascular devices and showed both measurable and visible splatter with a winged collection device.1 Studies since have taken this type of evaluation further looking at specific devices and the mechanism of activation.2

So, what does this tell us about the risks that phlebotomists and lab techs face every day from this potential for exposure that is often unrecognized? Because healthcare personnel must be aware of all avenues of exposure in order to take the necessary precautions, it is extremely important that use of safety devices meant to protect them does not create an additional risk of infection. Picking the safest device for use should not only include consideration of reduction in needlestick injuries but also assessment of splatter and review of studies such as those cited here to properly evaluate performance. Additionally, especially with devices associated with greater incidence of splatter, the appropriate protective equipment, e.g. face shields or googles, gowns, etc., should be utilized to prevent exposure and potential infection from bloodborne pathogens.

In summary, it is crucial that healthcare employees are equipped with the appropriate safety equipment when dealing with the potential for biohazardous exposure, that they understand the impact of the products being used and how these products can either help or hinder protection from bloodborne pathogens.

References

  1. Haiduven DJ, Applegarth SP, Shroff MP. (2009). An experimental method for detecting blood splatter from retractable phlebotomy and intravascular devices. Am J Infect Control 37(2); 127-130.
  2. Haiduven DJ, McGuire-Wolfe C, Applegarth SP. (2012). Contribution of a winged phlebotomy device design to blood splatter. Infect Control Hosp Epidemiol 33(11); 1069-1076.

-Michelle McLean, MS, MT(ASCP), BS is currently the Scientific Affairs Manager for Greiner Bio-One Preanalytics in North America. In this role, she is responsible for new product development and associated clinical studies, defining appropriate device application and developing technical and educational materials to support the preanalytic product portfolio. She is a Medical Technologist with an additional Bachelor of Science Degree in Biology and a Master of Science Degree in Pharmacology & Physiology. 

-Mackenzie Farone is a Senior Manager of Corporate Communications, Greiner Bio-One North America

Cytology Case Study: Strike a Chord

Every FNA ROSE attended where the patient is conscious and attentive can be tricky to navigate. You have to remain cognizant of your word choice, your demeanor, and the delivery of your adequacy statement to the clinician. The patient is already in a heightened state of awareness because he or she is about to be probed with a needle (or six!) for a test that is likely to rule out a benign or malignant process. I prefer to go into my biopsies with some sort of clinical picture and as many details as I can retain – is there a previous history of cancer? Where is the lesion located? Is it a single mass or are there multiple lesions? What does the radiologic imaging suggest? Are there any elevated serum tumor markers? I need to be able to walk the walk and talk the talk. However, there are rare instances when cytotechs are asked to rush down to an unscheduled add-on biopsy where we have yet to research the impression documented in the patient’s medical record. In those situations, I ask the clinician (typically an interventional radiologist) all the questions I can think of while still emulating some form of confidence to the patient.

I entered the procedure room and greeted the radiologist, radiology fellow, tech, nurse, and the patient, a 57-year-old male who was prone and alert on the table. I jotted down notes during the timeout and pulled the radiologist aside to ask, “does the patient have a history of cancer?” In this case, the answer was “they have a soft tissue tumor in the left gluteus, which is what we’re biopsying.” “Alright, let’s get those differentials rolling – sarcoma; after my hibernoma experience – a lipomatous tumor; or could it be a carcinoma (because yes, I’ve seen a lung adenocarcinoma metastasize to the gluteal muscle before)? Hmm… what else? What other mesenchymal tumors could originate here… or metastasize here?” My brainstorming balloon was popped by the radiologist asking if I was ready for the first needle pass. I replied, “Yes, of course!” I glance over at the patient and smile, trying to assure him AND myself that I’ll be able to give him a definitive answer to his puzzle.

Here’s what I visualized under the microscope after I stained the first air-dried smear in our Diff-Quik solutions.

Images 1-2. Left gluteal FNA, DQ-stained smears.

My inner monologue became: “Well, it’s not a sarcoma or a carcinoma. It doesn’t look malignant.  Not quite a hibernoma. What is with that myxoid matrix? It’s not mucinous or serous, so… what is it…? It’s granular! Wait. Those nuclei. They’re so… what’s the word? It’s definitely representative of the lesion. Regardless, it’s adequate!” I turned away from my microscope to face the team – “The sample is adequate. May I have a few more passes for my cell block, and will you collect core biopsies, too? “Yes and yes,” the radiologist replied. I smiled again at the patient, and he mouthed, “thank you.” “Phew, mission accomplished,” I thought. “Now what the heck are those hallmark cells called mixed in with a majority of epithelioid cells arranged in chords?”

I climb the stairs up to the lab and do a quick Google search. “DUH! Physaliphorous cells!” These cells have a distinct feature where the nucleus is centrally located but is also scalloped by cytoplasmic vacuoles. There weren’t as many physaliphorous (physaliferous) cells as I had hoped to appreciate. Some of the cells looked lipoblastic in nature with larger vacuoles displacing the nuclei to the periphery, almost signet ring in nature, many were cuboidal. But that was it… those cells! Now, imagine the scene in Finding Nemo where Nemo struggles to tell his classmates he lives in an anemone. That was my garbled attempt at pronouncing “physaliphorous” to the attending pathologist when sharing my interpretation. She looked at me like I was saying anything other than the word I was trying to reproduce. I cannot blame her; I still turn beet-red at the memory. But I was convinced that a chordoma was the tumor I presented to her.

After I processed my FNA, I examined the patient’s electronic health record to see if he had any previously biopsied neoplasms on file, and much to my surprise, the patient was diagnosed with a primary chordoma of the sacrum and treated with en bloc resection and radiation in 2013. Mutation analysis was performed on the resection of this chordoma, which exhibited a homozygous loss of CDK2NA (p16). The patient had one recurrence at an outside facility in 2015 and transferred his care to our institution for follow-up. Now, the patient presented with this gluteal metastasis and soon thereafter, a paraspinal metastasis. As the patient’s chordoma did not completely respond to radiation, the clinical care team turned to the tyrosine kinase inhibitor, Gleevec, which was discontinued due to disease progression. The patient’s regimen then went on to include sunitinib, which was also discontinued due to disease progression, palbociclib, then nivolumab, followed by radiation to the thoracic spine, sorafenib, and now is on a clinical trial for patients with advanced refractory cancers.

When I turned in my Diff-Quik & Pap-stained slides and the cell block H&E sections with a diagnosis of chordoma the next day, the attending cytopathologist paged through one of our cytology texts to a tidbit on chordomas before signing out the case. She reviewed the section with me. Other than the unique physaliphorous cells, it turns out a diagnosis of chordoma is fairly rare, as it is the only malignancy derived from the notochord, typically occurring at either end of the axial skeleton.1 Metastasis of these tumors is also rare, so this case of widespread metastatic disease was even more intriguing to me.

Images 3-8. Left gluteal FNA . Images 3-5, Pap-stained smears; 6-8, H&E cell block sections.

References

  1. Cibas, E. S., & Ducatman, B. S. (2009). Cytology: Diagnostic Principles and Clinical Correlates, Expert Consult – Online and Print (3rd ed.). Saunders.

-Taryn Waraksa, MS, SCT(ASCP)CM, CT(IAC), has worked as a cytotechnologist at Fox Chase Cancer Center, in Philadelphia, Pennsylvania, since earning her master’s degree from Thomas Jefferson University in 2014. She is an ASCP board-certified Specialist in Cytotechnology with an additional certification by the International Academy of Cytology (IAC). She is also a 2020 ASCP 40 Under Forty Honoree.

What to Expect When You Don’t Know What You’re Expecting: COVID-19 and Flu Season in the Laboratory

Welcome to October 2020 and a flu season unlike any other. What can we expect? Well, it’s complicated. And if we aren’t sure what to expect, can we still be prepared? Yes (at least for some things)!

From the beginning of the COVID-19 pandemic and throughout the summer of 2020 clinicians and laboratorians have been anxiously wondering what effect global presence of respiratory virus SARS-CoV-2 would have on the 2020-2021 flu season. “Flu season,” the annual, relatively predictable period of increased cases and deaths due to Influenza A and B, occurs during colder, winter months. In the northern hemisphere this is September through March. We have extensive experience tracking the onset and genetic variability of the predominant influenza viruses. We manufacturer flu vaccines based on data of potentially likely influenza strains. Other viruses that cause respiratory symptoms follow similar seasonal patterns. These include common (non-SARS-CoV-2) human coronaviruses, and Respiratory Syncytial Virus (RSV). In short: this is a known, annual occurrence that we can usually prepare to some extent.

So what will that look like this year? During the historic 1918 pandemic influenza, deaths seen during the first winter of the outbreak paled in comparison to those seen the following winter. Even if that kind of terrible scenario doesn’t occur during this pandemic year, it is possible we are facing “perfect storm” of COVID-19 plus influenza resulting in overwhelmed hospitals and depleted testing supplies. [https://www.cidrap.umn.edu/news-perspective/2020/09/fears-perfect-storm-flu-season-nears]

We know that COVID-19 spreads well in enclosed spaces with prolonged person-to-person contact, regardless of climate and temperature, via respiratory secretions. Because of this, there has been widespread adoption of mask wearing, social distancing, and limitations on in-person gathering. Promisingly, these interventions to prevent the spread of COVID-19 seem to be contributing to historically low influenza rates in the Southern Hemisphere! [https://www.cdc.gov/mmwr/volumes/69/wr/mm6937a6.htm] But adoption of these mitigation strategies are not being universally or rigorously followed in all regions and communities. As temperatures drop, we could see more people conducting activity indoors – will this change transmission patterns? Will regions with ongoing COVID-19 outbreaks be more prone to influenza as well? If hospital capacity becomes strained, will criteria for ordering tests change?

During COVID-19 laboratories have responded heroically and rapidly to test kit shortages, supply chain issues, and staffing challenges. At this stage (October of 2020) many high-level decisions about SARS-CoV-2 testing, like test platform purchasing and validation or manufacturer test kit allocations, might already be set in stone. So is there anything that can be done to help labs and laboratory workers successfully make it through flu season?

Here are 3 suggestions:

1) Establish testing algorithms and clear sample workflows.

Each facility and laboratory will have their own platforms for testing COVID-19 and other respiratory pathogens. Depending on the service ordering the test, there can be both immediate and downstream consequences for when a test comes back positive, negative, or even when that test result is slower than expected!

An algorithm helps set institutional expectations for what tests are ordered under different scenarios. For example symptomatic patients presenting to a hospital with influenza-like illness (ILI), especially when they will be admitted, should likely have both SARS-CoV-2 and influenza tests ordered simultaneously. But asymptomatic patients being admitted for procedures may only require a SARS-CoV-2 test.

Let’s say your lab has both a SARS-CoV-2 PCR test and SARS-CoV-2 rapid antigen test. But due to risk a false negative, lab and clinical leaders are uncomfortable using only a rapid antigen test to conclusively rule out COVID-19 in patients being admitted to the hospital. Your algorithm could use specify the use of SARS-CoV-2 antigen testing in symptomatic patients to quickly “rule in” potential positives, where antigen-negative patients will also have a PCR test. Algorithm specifics come down to what your institutions stake holders (clinical AND laboratory) need and capacity are. The details of an algorithm will be dependent on your lab test platforms, your available test orders, and may need to be modified to accommodate restricted test allocations.

Along with clinical algorithms, clear workflow for specimens and test types can help laboratory workers get tests where they need to go within the lab. Not all SARS-CoV-2 tests have approval in the instructions for use for, say, nasal swabs. If nasal swab comes to the lab with orders for both influenza and SARS-CoV-2 tests, what is the procedure for informing the floor for an appropriate collection? Or say that your test platforms for different tests live in different areas of the lab. Your workflow may be to set up one test and do a pour off into an aliquot tube so tests can be run at the same time. Or you may have sufficient test collection materials to request a separate sample for each test.

Probably the most important part of developing or reviewing your existing algorithms and laboratory workflow is doing it in connection with others. The purpose is to streamline the entire process from clinical decision making to test performing and reporting and help everyone be on the same page.

2) Communicate to clinical staff frequently about your tests.

Because of the intense interest surrounding COVID-19 laboratory testing, it’s entirely possible that more people have had to learn about previously niche laboratory concepts like “sensitivity vs. specificity” and “PCR vs. antibody vs. antigen tests” than at any previously time in human history! However, it is also likely that many clinicians or administrators in your own institution may know more about a test platform they read about in the news than the COVID-19 test platform that their laboratory performs.

Even at this stage in the pandemic with perhaps more exposure (pun not intended!) then the laboratory has ever had, miscommunication and unclear expectations abound surrounding test performance or turnaround times.

Whenever possible, lab leaders who interact with clinicians and administrators should look for ways to educate on test platforms, testing capacity, and expected test performance (i.e. time to result, comparative sensitivity etc.). This could include asking for time to provide formal updates during monthly meetings, monitoring test statistics (e.g. a test “dashboard”), or just informal reminders about what tests the lab performs during phone calls.

3) Keep the lab staff off the phone.

A critical part of the job of the lab is to provide information and updates on when test results are available. But when the hospital floors or clinics are busiest with patients, often the lab is busiest performing those patients’ tests. A phone call about the status of a respiratory virus test can be undeniably helpful to that patient’s clinical care team! But a dozen such phone calls over the course of a lab worker’s shift, especially under normal lab conditions (e.g. no staff shortages or instrument issues) is a failure of communication and can be detrimental to both lab performance and lab worker wellbeing.

In addition to the need for regular education about testing mentioned above, to help protect your lab staff’s bench time here are some possible ways keep from being overwhelmed with phone calls:

  • In some institutions, passive reminders (for example about hand hygiene or upcoming events) cycle through computer screen savers or on television screens in clinical areas. You could see if a message like “Reminder from the lab: COVID-19 tests are completed in [length of time].” could be put on a rotation.
  • If there is no client service or switchboard for your lab, but people call the lab directly for updates, you could institute a message stop. This is where phone calls routed to the laboratory must listen to a reminder that (for example), “If you are calling for an update of a COVID-19 test, these tests cannot be completed faster than [length of time] after arriving in the lab.”

    While these messages can be undeniably annoying and disruptive for people calling the lab for other reasons (and become less effective over time) if phone calls get out of hand, this option could be considered.
  • A lab instrument going down can result in test backlogs and numerous phone calls to the lab. Some institutions centralize their information in the form of a duty officer (for example in the emergency department). This will be a person who can be informed of actionable information, like test delays due to instrument issues, and who will post and distribute that information to those affected.

There is a lot we don’t know about what’s to come in the COVID-19 pandemic. While we can’t predict the ways the lab may be challenged with the next unforeseen disruption, or even what our flu season testing needs may look like, hopefully we can prepare now to continue to support our patients by helping and supporting our labs.

-Dr. Richard Davis, PhD, D(ABMM), MLS(ASCP)CM is a clinical microbiologist and regional director of microbiology for Providence Health Care in Eastern Washington. A certified medical laboratory scientist, he received his PhD studying the tropical parasite Leishmania. He transitioned back to laboratory medicine (though he still loves parasites!), and completed a clinical microbiology fellowship at the University of Utah/ARUP Laboratories in Utah before accepting his current position. He is a 2020 ASCP 40 Under Forty Honoree.

The Case for a Laboratory Quality Team

Maintaining a high level of quality is one of the primary goals of U.S. clinical laboratories. Sustaining quality is especially crucial during the often tumultuous and seemingly uncertain times that accompany hospital and healthcare systems mergers. According to an analysis by Kaufman Hall, there were 27 hospital and health systems mergers in the first quarter of 2019, representing revenue of $4.9 billion.  Mergers, acquisitions, and growth can be disruptive activities affecting finances, daily operations, and employee morale.

The majority of hospitals and healthcare delivery organizations have a quality department. However, the department is more often focused on the tasks and activities occurring outside of the laboratory. Furthermore, many hospital quality departments consist mainly of nursing or other clinical staff types and lack laboratory representation.

Often, despite the importance of test data, the laboratory’s role in a hospital’s quality department is usually limited.  If the laboratory is involved, it is frequently because a test result may have been involved in an event. Few hospital quality departments directly employ laboratory professionals.

Clinical laboratories must adapt to healthcare organization changes while simultaneously continuing to meet regulatory and accrediting standards. How can a laboratory ensure the changes do not affect the quality and remain “inspection-ready?” How does a large healthcare system standardize quality across multiple laboratories? The answer is a Laboratory Quality Team.

Laboratories are complex and highly regulated operations that must meet CLIA regulations and specific accrediting standards. A laboratory quality team comprised of seasoned laboratory professionals provides the healthcare organization with in-depth laboratory operations experience, real-world quality control knowledge, and practical understanding of regulatory and accrediting standards.

Another great benefit of a laboratory quality team is that their primary focus is on monitoring and maintaining quality in the laboratory. This benefit includes the ability to conduct root cause analyses (RCAs), reviewing and ensuring validation of new tests and instrumentation, and conducting internal audits.

The value of having a dedicated laboratory quality team is readily apparent in mergers, especially when one of the goals of the merger is to streamline processes, standardize operations, and reduce waste.  A laboratory quality team is a perfect tool to conduct a gap analysis between different laboratories to determine the best and most cost-effective processes and procedures to retain.

A dedicated laboratory quality team is an invaluable asset for a healthcare organization operating multiple labs (particularly those in different states). The team can organize and coordinate quality-related activities and performance improvement initiatives. Assessing the level of compliance across laboratories and preparing compliance-related reports would be a primary responsibility of these individuals. The team would also be directly involved in developing, revising, and validating laboratory quality administrative policies and procedures.

As an outcome of new knowledge and scientific breakthroughs, laboratory accrediting bodies are continuously updating standards governing laboratory operations. A laboratory quality team can research and provide clarity on regulatory mandates and accrediting standards. The team can also be a resource on how changes in standards may affect laboratory operations and evaluate whether current procedures or processes need to be updated or eliminated.

Customer service is an essential aspect of laboratory operations. Beyond patients, laboratories serve physicians, nurses, and other healthcare providers. The laboratory quality team can represent the lab when interfacing and collaborating with leadership, interacting with medical and laboratory staff, and is especially suited to interact with lab inspectors.

The laboratory quality team’s minimum qualification should be a bachelor’s degree in a physical, chemical, biological science or medical technology and at least two years of clinical lab experience. However, a more robust set of qualifications would include experience as a clinical laboratory inspector, additional auditor certification (for example, those offered by the American Society of Quality), and personality and skills geared toward researching, learning, and presenting.

Mergers, consolidations, and acquisitions will continue in the healthcare industry as the marketplace environment challenges organizations’ bottom-line. Laboratories are an integral part of hospitals and other healthcare organizations seeking to deliver high-quality services to the community. Lab administrators and managers need to adapt operations while sustaining a high level of quality. A laboratory quality team composed of seasoned, knowledgeable, and motivated professionals can help laboratories navigate the inevitable regulatory, accrediting, and personnel changes unavoidable in the dynamic healthcare industry.

-Darryl Elzie, PsyD, MHA, MT(ASCP), CQA(ASQ), has been an ASCP Medical Technologist for over 30 years and has been performing CAP inspections for 15+ years. Dr. Elzie provides laboratory quality oversight for four hospitals, one ambulatory care center, and supports laboratory quality initiatives throughout the Sentara Healthcare system.

Microbiology Case Study: A Middle-Aged Woman with Forearm Pain

Case History

A middle-aged female was evaluated for left forearm pain and erythema following a cat bite one-day prior, and was prescribed trimethoprim-sulfamethoxazole for management in the outpatient setting.  She subsequently presented for follow-up where she was noted to have a 3 x 4 cm raised, red, indurated lesion of left arm without any discharge (Image 1).  MRI demonstrated a 6.5 x 2.3 x 2.3 cm abscess within the distal ulnar soft tissues with surrounding cellulitis.  As her pain and erythema were progressively worsening, she was admitted for surgical management.   

Upon admission, a bedside incision found purulent drainage which grew mixed anaerobic gram negative rods.  Blood cultures collected at this same time were negative and remained so for the duration of her hospital course.  Empiric antibiotic therapy was initiated with piperacillin-tazobactam, and the patient underwent formal surgical incision and drainage.  Intraoperative findings were notable for abscess, diffuse and severe tendinopathy, and a thick inflammatory rind surrounding the associated neurovascular bundle.  Intraoperative cultures were obtained and sent to the microbiology laboratory.  The patient’s postoperative course was uneventful, and she was discharged with plans to complete a two week course of amoxicillin-clavulanate.  Follow-up clinic visits demonstrated successful recovery, with a well-healed incision and normal grip strength and range of motion.

Laboratory Identification

Bacterial culture of abscess material collected intraoperatively grew smooth, mucoid colonies on chocolate and blood agars with less than 24 hours of incubation at 35°C in CO2 (Image 2, bacterial isolate). Growth was notably absent on MacConkey agar. Gram stain of the colony revealed tiny, gram negative coccobacilli (Image 2). Biochemical testing determined this organism to be indole, oxidase, and catalase positive.  The organism was definitively identified as Pasteurella multocida by MALDI-TOF MS.

Image 1. Arm lesion prior to incision and drainage.
Image 2. P. multocida growth on Blood and Chocolate agars. Gram stain from a colony revealed small, gram negative coccobacilli (far right).

Discussion

Members of the genus Pasteurella are small, Gram-negative coccobacilli which are able to readily grow on Sheep’s blood agar and chocolate agar, but will typically not grow on MacConkey media.  Infection with these organisms is usually considered to be a zoonosis, with both wild and domestic animals serving as reservoirs.  In animal hosts they can be part of the endogenous flora or pathogens.  P. multocida is the most common member of the genus associated with human infections, which has now been divided into multiple taxonomic subspecies through the use of more discriminatory molecular methods.  Biochemically, P. multocida is positive for catalase, oxidase, indole, and nitrate reduction.

Animal bite wounds are often polymicrobial and contain mixtures of both aerobic and anaerobic organisms.  These organisms can be reflective of the oral flora of the biting animal, or of endogenous skin flora of the bite victim.1  While 80-90% of bites per year can be attributed to dogs, an estimated 400,000 cat bites (5-10% of the total) occur in the United States annually.2  While dog bites often manifest as localized crush injuries with contusions and/or lacerations, a majority of such wounds are accessible to irrigation and cleaning which leads to a relatively low infection rate (5-10%).  By contrast, cat bites are often deep, localized puncture wounds which provide excellent environments for the growth of both aerobic and anaerobic bacteria.  While feline bite wounds may appear less severe after cursory inspection, these wounds can be considerably more difficult to clean, resulting in overall infection rates up to 50%.3 

Management of bite wounds includes cleansing, irrigation and debridement.  Importantly, antimicrobial therapy should include coverage for both aerobic and anaerobic bacteria.4  In this case, amoxicillin-clavulanate was utilized with good results, and provides coverage for the most common oral aerobes and anaerobes encountered in animal bite wounds.  Amoxicillin-clavulanate also has activity against beta-lactamase producing bacteria such as Prevotella sp. and Porphyromonas sp. which are oral anaerobes of dogs, cats, and humans.  The use of macrolides should be avoided due to variable activity against Pasteurella multocida.4  As in this case, bite wounds most frequently are encountered on the upper extremities, and Pasteurella sp. is one of the most common isolates recovered from bites from both cats and dogs (50% of dog bites, and 75% of cat bites).2

References

1. Abrahamian FM, Goldstein EJC. 2011. Microbiology of Animal Bite Wound Infections. Clinical Microbiology Reviews 24:231.

2. Bula-Rudas FJ, Olcott JL. 2018. Human and Animal Bites. Pediatrics in Review 39:490.

3. Kannikeswaran N, Kamat D. 2008. Mammalian Bites. Clinical Pediatrics 48:145-148.

4. Stevens DL, Bisno AL, Chambers HF, Dellinger EP, Goldstein EJC, Gorbach SL, Hirschmann JV, Kaplan SL, Montoya JG, Wade JC. 2014. Practice Guidelines for the Diagnosis and Management of Skin and Soft Tissue Infections: 2014 Update by the Infectious Diseases Society of America. Clinical Infectious Diseases 59:e10-e52.

 -Francesca Lee, MD, is an associate professor in the Departments of Pathology and Internal Medicine (Infectious Diseases) at UT Southwestern Medical Center.

-Huy Dao, MLS(ASCP)CM graduated from the University of Minnesota and has worked for eight years as medical technologist for eight years.  He is interested in clinical mycology and bacteriology.

-Andrew Clark, PhD, D(ABMM) is an Assistant Professor at UT Southwestern Medical Center in the Department of Pathology, and Associate Director of the Clements University Hospital microbiology laboratory. He completed a CPEP-accredited postdoctoral fellowship in Medical and Public Health Microbiology at National Institutes of Health, and is interested in antimicrobial susceptibility and anaerobe pathophysiology.

Monitoring Bone Marrow Transplant Recipients

Hello everyone, it’s been quite some time since my last post. I hope everyone has remained safe and healthy during these times!

My last post dived into short tandem repeat (STR) analysis for bone marrow engraftment monitoring. Today is a presentation of a patient who was transplanted for treatment of acute myeloid leukemia (AML). With all patients (with minor exceptions), donor and pre-transplant recipient samples are taken before transplant. Their informative alleles are then identified and used to determine the percent of donor and any recipient cells in subsequent post-transplant samples.

This patient was unique in that we were not able to obtain the donor sample (they were transplanted outside of our system), and therefore we used a buccal swab for their pre-transplant recipient informatives.

Buccal swabs are chosen because they are a non-invasive way to obtain squamous epithelial cells. These cells are important because they are of the recipient origin and will not change. With this technique, it is essential that the patient has no mucosal inflammation or is not too rough when swabbing their cheek. Otherwise, the buccal sample may become contaminated with blood which would contain donor cells.

We then inferred the donor informatives from the data of a mixed sample and the buccal swab.

Calculation of recipient and donor percentage in a post-transplant sample is determined on specific formulas that utilize these informative alleles. But what happens when a patient relapses and new mutations or deletions are introduced into their genome, causing a change in these informative alleles?

In this case, the patient had a loss of allele at two loci (CSF1PO – allele 11 and D5S818 – allele 13) after having previously obtained full engraftment (Figure 1).

Figure 1. The pre-sample was acquired through a buccal swab. There was no donor sample that was acquirable, and therefore the donor informative alleles were inferred through available data. In September of 2019, the patient was at 100% donor. Almost a year later, the patient is now at 4% donor and missing previously identified recipient alleles, indicating a loss of allele/mutation. Brown box with “R” stands for recipient. Blue box with “D1” stands for donor. Green box with D1R stands for shared.

The importance here is that the true percent donor is 4% (Figure 2). If we take a look at the affected informative alleles, we see an erroneous result of 100% donor and NI (which means the locus is non-informative, eliminating it from the calculations). This expands on the importance of an analyst to be attentive to the results presented. While this case was clearly evident and was caught by our error measurements, it is theoretically possible to cause an issue, especially in cases where the recipient percentages may be smaller. Furthermore, this phenomenon stresses the importance of including multiple informative alleles in our analysis, which increases our measurement of confidence.1

Figure 2. CSF1PO and D5S818 are incorrectly representing the patient’s status. CSF1PO is representing the patient at 100% donor and D5S818 is automatically identified as a non-informative by our software. After automatic and manual loci ignores, the total percent donor was 4%

We know that a loss of allele (loss of heterozygosity) is the likely explanation because both loci are in locations specific to the disease. Looking at Figure 3 below, the two alleles were affected because they were both present on the long arm of chromosome 5. Further, this chromosome is known to be involved in AML, and is also, of course, associated with other disorders like MDS.2 Additionally, the patient had cytology testing that identified this as an affected chromosome.

Figure 3. CSF1PO and D5S818 are both located on the long arm of chromosome 5. CSF1PO’s location is 5q33.1 and D5S818’s location is 5q23.2.

This is an interesting phenomenon and one that shows in measurable terms how a patient’s status can affect their molecular results. It’s further an expression of the molecular mechanisms of a disease, one of my first measurable experiences of how a disease affects the physical molecular constituents of another human.

To me, this encounter was an expression of how complicated, and yet connected, the entire genome has been designed. I am continuously amazed and look forward to expanding my understanding of molecular science.

References

  1. Crow J, Youens K, Michalowski S, et al. Donor cell leukemia in umbilical cord blood transplant patients: a case study and literature review highlighting the importance of molecular engraftment analysis. J Mol Diagn. 2010;12(4):530-537. doi:10.2353/jmoldx.2010.090215
  2. Crow J, Youens K, Michalowski S, et al. Donor cell leukemia in umbilical cord blood transplant patients: a case study and literature review highlighting the importance of molecular engraftment analysis. J Mol Diagn. 2010;12(4):530-537. doi:10.2353/jmoldx.2010.090215

-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: 83 Year Old Female with a Perisplenic Abscess

Case History

An 83 year old female with a past medical history of breast cancer, multiple strokes, dysphagia, hypertension and gastroesophageal reflux disease (GERD) presented to an outside hospital with altered mental status, metabolic encephalopathy, decreased appetite, acute kidney injury, and E. coli/Proteus urinary tract infection (UTI). There, she was diagnosed with a perforated gastric ulcer, which was repaired, with a gastrostomy (G) tube in place. The patient later developed a fever and an aspiration pneumonia, which was treated with ampicillin/sulbactam. A follow up imaging revealed a new gastric perforation along the fundus of the stomach with perisplenic fluid collection, along with a pleural effusion with possible communication with the fluid collection in the stomach. Due to her complex medical history, an additional intervention was not pursued and the family agreed to comfort measures, and the patient was discharged home.

The family presented to our emergency department the same day of discharge, as the patient had not been eating and the family needed assistance in using the G tube. In-house computed tomography (CT) of the abdomen/pelvis again showed an organizing collection near the spleen (Image 1). A medium-sized left pleural effusion with left lower lobe collapse due to the communication with the perforation was observed on CT. The patient received IV fluids and a dose of vancomycin and zosyn in the ED. A vascular and interventional radiology (VIR) consult was recommended for potential drainage of the perisplenic abscess and left pleural effusion.

Image 1. CT of the abdomen demonstrating an organizing collection (black circle, approximately 7 cm x 3 cm) posterior/superior to the spleen near the site of the prior gastric perforation concerning for an abscess.
Image 2. Small gram negative rods on a Gram stain of Burkholderia cenocepacia from a subculture.
Image 3. Culture morphology of Burkholderia cenocepacia on a blood agar plate after overnight incubation: smooth colonies are present (upon prolonged incubation, the colonies turned yellowish color – not shown in image).

VIR drained the perisplenic abscess, which was sent to the microbiology lab for aerobic & anaerobic cultures. The Gram stain revealed many white blood cells (WBC’s) and rare yeast. The culture grew 3+ Burkholderia cepacia complex (Burkholderia cenocepacia) and 3+ Candida glabrata. The Gram stain and colony of the subculture of B. cepacia on a blood agar plate are shown in Images 2 and 3. C. glabrata was also isolated from the urine culture. Susceptibility testing of B. cepacia showed that it was sensitive to both meropenem and trimethoprim-sulfamethoxazole. Vancomycin and zosyn were stopped and the patient was switched to IV trimethoprim sulfamethoxazole for B. cepacia and IV micafungin for C. glabrata.

Due to the recurrent perforation, the G tube could not be used; a jejunostomy (J) tube was placed instead. Feeds were successfully started with the J tube. Trimethoprim-sulfamethoxazole was also given via the J-tube. A follow-up endoscopy showed a normal esophagus, the known perforation in the gastric fundus, and erythematous duodenopathy at the level of the duodenal bulb, the remainder of the duodenum was normal. The patient’s clinical status improved and she was discharged home with the support of home health services.

Discussion

We present an uncommon presentation of Burkholderia cenocepacia, a member of B. cepacia complex, in perisplenic abscess fluid. B. cepacia complex consists of at least 17 closely related species. They are rod-shaped, aerobic, motile Gram-negative bacteria. B. cepacia complex has been well characterized as opportunistic pathogens, particularly in patients with cystic fibrosis and chronic granulomatous disease (CGD). However, they can also infect immunocompetent patients and have been reported to cause endocarditis (specifically in IV drug abusers), pneumonitis, UTIs, osteomyelitis, dermatitis, and other wound infections. In the United States, B. multivorans and B. cenocepacia together account for approximately 80% of B. cepacia complex infections. Burkholderia have been isolated on contaminated hospital equipment and even disinfectants. They present a large problem in nosocomial infections due largely to their ability to survive in aqueous environments.1 They are soil-dwelling bacteria commonly found on plant roots. They are of environmental interest secondary to their antifungal and antinematodal properties as well as the ability to degrade many toxic compounds in agriculture (pesticides, herbicides, preservatives).2 Clinically important Burkholderia species outside of the B. cepacia complex include B. pseudomallei, the causative agent of melioidosis, and B. mallei, the causative agent of glanders.1

Rare case reports have previously documented B. cepacia isolated from splenic abscesses/infections. Most B. cepacia splenic infections occurred secondary to pneumonia or multi-organ involvements in CGD patients. 3, 4, 5 However, one report indicated the B. cepacia-mediated multiple splenic abscesses, in the setting of malignancy and diabetes. 6 While the splenic abscesses in the context of meliodosis, either due to B. pesudomallei or B. mallei infection, have been reported, 7 B. cepacia-mediated splenic infections are rarely encountered.

B. cepcacia complex has intrinsic resistance toseveral antibiotics including penicillins, amoxicillin-clavulanate, ertapenem, polymixin B, Colistin, and fosfomycin.8 B. cepacia complex possesses an inducible β-lactamase, encoded by the gene penA, which can hydrolyze penicillin and use it as a source of carbon. In one study involving 40 bloodstream isolates of B. cepacia in patients without cystic fibrosis, 93% of the isolates were susceptible to ceftazadime and 95% of isolates were susceptible to trimethoprim-sulfamethoxazole.9 Following discussion with our infectious disease colleagues, we believe that the B. cepacia isolate from our patient was likely a nosocomial infection from possible contamination of her G-tube in combination with the gastric perforation.

References

  1. Jorgensen, J. H., Pfaller, M. A., & Carroll, K. C. (2015). Manual of clinical microbiology. Washington, DC, DC: ASM Press.
  2. Kenyon College Department of Biology. (2011, April 22). Burkholderia cepacia. Retrieved September 21, 2020, from https://microbewiki.kenyon.edu/index.php/MicrobeWiki
  3. Clegg HW, Ephros M, Newburger PE. Pseudomonas cepacia pneumonia in chronic granulomatous disease. Pediatr Infect Dis. 1986 Jan-Feb;5(1):111. PMID: 3945563.
  4. Sirinavin, Sayomporn MD*; Techasaensiri, Chonnamet MD*; Pakakasama, Samart MD*; Vorachit, Malai DSc; Pornkul, Rattanaporn MD; Wacharasin, Rames MD Hemophagocytic Syndrome and Burkholderia cepacia Splenic Microabscesses in a Child With Chronic Granulomatous Disease, The Pediatric Infectious Disease Journal: September 2004 – Volume 23 – Issue 9 – p 882-884 doi: 10.1097/01.inf.0000137565.23501.03
  5. Bottone EJ, Douglas SD, Rausen AR, Keusch GT. Association of Pseudomonas cepacia with chronic granulomatous disease. J Clin Microbiol. 1975 May;1(5):425-8. doi: 10.1128/JCM.1.5.425-428.1975. PMID: 1176612; PMCID: PMC275137.
  6. Jayawardena, M. N., Chandrasiri, N. S., Wijekoon, S., Madanayake, P., Corea, E., Ranasinghe, D. D., & Lamahewage, N. D. (2017). Burkholderia cepacia; an unusual cause of multiple splenic abscesses : A case report. Sri Lankan Journal of Infectious Diseases, 7(2), 123. doi:10.4038/sljid.v7i2.8146
  7. Chen, H., Hu, Z., Fang, Y., Lu, X., Li, L., Li, Y, Mao, X, Qian, L. (2018). Splenic abscess caused by Burkholderia pseudomallei. Medicine, 97(26). doi:10.1097/md.0000000000011208
  8. Patel, J. B., Weinstein, M. P., Eliopoulos, G.M., Jenkins, S.G., Lewis, J.S., Limbago, B., Mathers, A., Mazzulli, T., Patel, R., Richter, S.S., Satlin, M., Swenson, J.M., Traczewski, M.M., Turnidge, J.D. & Zimmer, B.L. (2017). Performance standards for antimicrobial susceptibility testing. Wayne, PA: Clinical and Laboratory Standards Institute.
  9. Bressler A.M., Kaye K.S., LiPuma, J.J., Alexander, B.D., Moore, C.M., Reller, L.B. & Woods, C.W. Risk factors for Burkholderia cepacia complex bacteremia among intensive care unit patients without cystic fibrosis: A case-control study. Infect Control Hosp Epidemiol 2007; 28(8):951-8 doi : http://dx.doi.org/10.1086/519177

-J. Stephen Stalls, MD is a PGY-II pathology resident at the East Carolina University Department of Pathology and Laboratory Medicine. He plans to pursue hematopathology and molecular pathology fellowships, but also greatly enjoys his time in the microbiology lab. Outside of work, he enjoys playing the drums and going to concerts.

-Phyu Thwe, PhD, MLS(ASCP)CM is Technical Consultant/Technical Director of Clinical Microbiology Laboratory at Vidant Medical Center, Greenville, NC.

Pathology and Global Public Health

Recently, I interviewed for a subspecialty surgical pathology fellowship, and one of my interviewers posed a question to me: “What has pathology got to do with global health?” She asked me that question because  my resume highlights some of my global health-related activities and interests.

Pathology1 is the foundation upon which other specialties in medicine are situated. However, pathology as a specialty is seldom talked about or even referenced by other colleagues in other clinical specialties. One possible reason for this is that pathologists are often out of the perceptions of other clinicians.

That’s unfortunate, because the roles of pathologists in patient care cannot be overemphasized. Pathologists have a solid understanding of the pathophysiology of various diseases. We can identify when tissues are diseased or free from disease, and can also differentiate between various disease processes. We are also versed in the molecular and genetic basis of diseases. We also develop biomarkers to identify different disease processes. Through the assessment of various histopathologic, immunohistochemical analyses and morphologic features, we can also prognosticate various disease processes, a process that has become more effective with advances in molecular pathology. In addition, through our expertise, we serve as consultants to clinical teams to guide patient management.

However, beyond playing the above critical roles, pathologists can exert their influences by getting involved in public health and global health discussions, to influence policies that ultimately impact the outcome of patients. During the COVID-19 pandemic, the role of pathologists and laboratory professionals has become more critical in healthcare delivery.2 We play a huge role in the development of rapid and effective diagnostic assays, as well as influence the interpretation and delivery of timely test results.  In addition, through the conduct of autopsies on deceased patients, we have been able to describe some of the clinical and morphologic alterations associated with the SARS-CoV-2 (coronavirus).3

In spite of our important roles in the practice and delivery of medical services, it’s not common practice to have pathologists sit on major hospital boards, or participate in policy discussions that impact healthcare delivery. In addition, many low resource settings outside the United States still experience a shortage of effective laboratory services, with its attendant catastrophic effects on patient care.4 And even in the United States, the pathology workforce is gradually shrinking which could portend dire consequences for effective patient care delivery.5 In order to gain more traction to our specialty, it’s time for pathologists, to step out of our comfort zones and become more visible in the communities that we serve.

Pathology and laboratory services in many developing countries are currently suboptimal from a combination of scarcity of trained pathologists to sub-standard laboratory operations. Pathologists can step in to close this gap by developing collaborations that could foster partnerships in care delivery, training and research opportunities. I want to highly commend healthcare institutions that currently have dedicated pathology global health programs.6

This is a call to action for our specialty. If we really want to become more visible, relevant and attract some of the best talents to our specialty, then we should be ready to show that we bring so much more to the table than just peering into the microscope. The value of our pathology reports in the management of patients cannot be over emphasized. However, we must exert our relevance and expertise in healthcare discussions by stepping out into the communities that need us the most. A great place to start is getting involved with local public/global health-related work, including exploring opportunities, offered by the American Society for Clinical Pathology (ASCP) Center for Global Health.7

References

  1. https://www.rcpath.org/discover-pathology/what-is-pathology.html#:~:text=Doctors%20and%20scientists%20working%20in,patients%20with%20life%2Dthreatening%20conditions.
  2. https://www.uab.edu/news/health/item/11259-working-behind-the-scenes-uab-pathologists-play-key-role-in-fighting-coronavirus-pandemic
  3. Calabrese F, Pezzuto F, Fortarezza F, et al. Pulmonary pathology and COVID-19: lessons from autopsy. The experience of European Pulmonary Pathologists. Virchows Arch. 2020;477(3):359-372. doi:10.1007/s00428-020-02886-6
  4. Sayed S, Lukande R, Fleming KA. Providing Pathology Support in Low-Income Countries. J Glob Oncol. 2015;1(1):3-6. doi:10.1200/JGO.2015.000943
  5. Lundberg GD. How Many Pathologists Does the United States Need? JAMA Netw Open. 2019;2(5):e194308. doi:10.1001/jamanetworkopen.2019.4308
  6. https://www.massgeneral.org/pathology/global-health
  7. https://www.ascp.org/content/get-involved/center-for-global-health


-Evi Abada, MD, MS is a Resident Physician in anatomic and clinical pathology at the Wayne State University School of Medicine/Detroit Medical Center in Michigan. She earned her Masters of Science in International Health Policy and Management from Brandeis University in Massachusetts, and is a global health advocate. Dr. Abada has been appointed to serve on the ASCP’s Resident’s Council and was named one of ASCP’S 40 under Forty honorees for the year 2020. You can follow her on twitter @EviAbadaMD.

Virtually Amazing

Hello everyone and welcome back!

I’ve appreciated some amazing feedback from my previous post discussing how doctors can sometimes be patients too, and the challenges one might face in different roles within our health care system. Not only a challenge of roles, but those that struggle with invisible illness have unique perspectives on patient care.

That said, this month let me take a break from all the fun content found between cases, concepts, and trends in pathology and laboratory medicine, and celebrate our amazingly successful (and virtual) Annual ASCP Meeting!

Image 1. Just look at this virtual lobby! Set aside that in-person connectivity dissapointment and just appreciate the quality put into this visually! More of my oggling to come in further images…

It was awesome. But don’t just take my word for it, we’re all people of science here, right? So let’s do it by the numbers!

  • 133 educational sessions
  • 3 general sessions
  • 4 named lectures
  • 36 round table sessions which included topics like wellness, problem-solving, collaborative solutions, and “birds of a feather” breakout discussions
  • 9 virtual video microscopy sessions
  • 8 session dedicated to laboratory professionals covering hematology, chemistry, microbiology, and blood banking
  • 6 resident board review sessions
  • 15 companion society sessions
  • 14 sessions related to wellness
  • 4 sessions discussing diversity and inclusion
  • 10 COVID-focused sessions
  • 20 grant funded sessions
  • 4 virtual patient symposia (more on this topic below…) and
  • And 300+ posters!
Image 2. More visual appreciation here: virtual sessions felt like you were really in a large, collective meeting of enthusiastic, like-minded laboratory professionals all learning, collaborating, and networking together!
Image 3. I was fortunate enough to to speak on this amazing panel regarding direct patient-and-pathologist interactions, making laboratory medicine and the overal healthcare experience, safer, more accessible, more interdisciplinary, and better equiped at dealing with the forefront of medical diagnostics!
Image 4. So, the session went well! Just look at that social media data: 36 million impressions over 3.5 days! That’s 1 million people engaging ASCP topics a day, or 12 people per second! All actively discussing and collaborating topics in pathology and laboratory medicine.
Image 5. How could I (of all people) ignore the fact that #ASCP2020 featured an amazing social (media) lounge where people from all over could connect, chat, network, and relax! There were interactive, virtual sessions covering all kinds of non-lab med stuff: yoga, meditation, mixology, and cooking! I hope this is a permanent addition to future (hopefully) hybrid in-person/virtual meetings.

What more could you ask for? The folks that run the logistics and planning for the ASCP Annual Meeting outdid themselves again. Sure this content would excite anyone in the field for 3 dedicated days of immersive learning and networking, but all this and more are still available online for virtual on-demand recorded viewing! Missed a session? No worries, it’s still waiting for you for about 6 months (through March of 2021). All the buzz aside from ASCP members having free access to all of this content, the excitement started months before the meeting went live. Estimates are still coming in, but membership grew by a couple hundred in the weeks leading up to the meeting—not surprising: free access for members? That was an excellent deal, so choice.

Image 6. The start of the #ASCPSoMeTeam’s amazing trajectory culminated at #ASCP2019 in Arizona, the more we work together the more we can accomplish for our profession and our patients, #StrongerTogether.
Image 7. ASCP’s Resident & Pathologist Councils are invaluable assets to promoting and advancing all of our professional development. #ASCP2020 was no different! From virtual fellowship fairs to online, interactive resident council sessions, there was a lot to take it—still available online!
Image 8. I’ve talked about previous ASCP Annual Meetings here and here, and while I can’t list every single aspect of what made this meeting (virtually) amazing, members can check in for about 6 months and see for themselves the quality and attention to detail that comes directly from our collective passion to make pathology and laboratory medicine better, for everyone. Kudos to the ASCP leadership and logistics teams that made this all possible!

Great to see you all at the meeting!

Thanks for reading! See you next time!


-Constantine E. Kanakis MD, MSc, MLS (ASCP)CM is a new first year resident physician in the Pathology and Laboratory Medicine Department at Loyola University Medical Center in Chicago with interests in hematopathology, transfusion medicine, bioethics, public health, and graphic medicine. His posts focus on the broader issues important to the practice of clinical laboratory medicine and their applications to global/public health, outreach/education, and advancing medical science. He is actively involved in public health and education, advocating for visibility and advancement of pathology and lab medicine. Watch his TEDx talk entitled “Unrecognizable Medicine” and follow him on Twitter @CEKanakisMD.

Case Study Hematology: The Mouse Strikes again! Lymphocytes with Intracytoplasmic Inclusions

If you read my last blog, you heard the about the story “if You Give a Mouse a Cookie” by Laura Numeroff.4 The curious little mouse has a mind that never rests. As his mind wanders and hops from one thing to another, he keeps discovering more things to check out along the way. Medical laboratory lcientists are a lot like this. We’re a curious bunch, and, in investigations, one thing often leads to the next. Well folks, the mouse has struck again! We were given another cookie in the form of these beautiful cells.

Image 1. Lymphocytes with intracytoplasmic inclusions.

These cells were found by my coworker Liz Marr, MLS(ASCP), and the adventure began! First, we wanted to know what those were, and then we needed to find out more about them, and then, mostly, I wanted to know why in almost 40 years of working in and teaching hematology that I have never before seen this!

The story begins with our case history. We received a CBC from a 71 year old female with a 4 year history of untreated chronic lymphocytic leukemia/ small lymphocytic lymphoma(CLL/SLL). The patient’s recent history included a myocardial infarction(MI) 5 months prior. The patient was found to have leukocytosis (WBC 25.38 x 103/μL) and absolute lymphocytosis (18.25 x 103/μL) with normal hemoglobin and hematocrit (Hgb 13.4 g/dL, Hct 40.8%) and normal platelet count (272 x 103/μL). The differential had 71.9% lymphocytes with many abnormal forms noted. The slide was sent for a pathology review. The pathologist reported “Atypical lymphocytosis consistent with patient’s known chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) Filament-like inclusions are present in the cytoplasm which has been previously reported in patients with CLL.”

Image 2. Lymphocytes with crystalline-like inclusions.

A curious tech can’t stop at just that description. If you tell me they are filament-like inclusions, I will have all kinds of questions. What are these filaments made of? Are they crystals, or something else? How common are these? Are these diagnostic of CLL? Are these only seen in CLL? What is their significance? And, of course, the most puzzling question, why have I never seen these before??

CLL is a form of non-Hodgkin lymphoma and is the most common leukemia in the Western world. It is generally a leukemia of older age with a median age at diagnosis around 67-72. The disease is widely variable, with some patients asymptomatic and requiring no treatment for many years, while others have a more rapidly progressive course of disease requiring treatment. About 60% of patients are diagnosed before they exhibit any symptoms. CLL and SLL are considered to be different manifestations of the same disease. In CLL, the abnormal B lymphocytes are found mostly in the peripheral blood and bone marrow, but in SLL, there is lymph node involvement, with abnormal cells mostly found in the lymph nodes. CLL is diagnosed based on absolute B lymphocyte counts ≥5 x 109/L. Flow cytometry typically reveals a distinctive cell immunophenotype with expression of CD19, CD5, CD23, and Κ/λ; and weak expression of CD20, CD79b, and surface immunoglobulin.1

The most recent flow cytometry report on our patient was from one year ago. An 8 color analysis with CD45/SSC gating was performed by LabCorp. The flow revealed an abnormal cell population representing 56% of total cells. Two monoclonal B cell populations were detected with identical phenotypes except for light chain expression. These cells expressed CD45, CD19, CD20, CD22, CD5, and CD23., CD38-. This phenotype was consistent with her previous diagnosis of CLL/SLL.

A literature search revealed only a few articles about intracytoplasmic inclusions in CLL. Cytoplasmic inclusions in lymphomas are uncommon, but have been noted as vacuoles, crystals, and pseudocrystals. These crystalline inclusions represent immunoglobulin(Ig) heavy and light chain that precipitate in the cytoplasm. Using electron microscopy it has been found that theses Ig deposits localize in the rough endoplasmic reticulum (RER).5 When surface Ig can be demonstrated on the B lymphocytes, it has been found to be same as Ig in the inclusions.6

In two published studies that describe these crystal like inclusions, photographs are very similar to the ones we found on our patient.3,5 It is interesting to note that, in these two studies, neither of the subjects was a known CLL patient. The inclusions were noted in the patients’ cells and the peripheral blood was subsequently sent for flow. Phenotypes reported confirmed monoclonal B-cells representing a large percentage of cells. Huang reported monoclonal B-cells which expressed CD45, CD19, CD20, CD22, CD79b, CD5, CD23, CD148 and CD200(hi), with partial expression lambda, and negative for FMC7, CD10, CD11c, CD49d, CD103, CD38, CD25, CD160, IgM, CD81, kappa and Ki67.3 In the Ramlal case study, phenotype was CD5, CD19, CD20, CD23, positive, CD10, FMC7 negative.5 On the basis of flow, along with the CBC results, the patients were diagnosed with CLL.

Of course, while researching this, the little mouse in me kept asking questions and finding more questions to ask. One question that I still had questions about was if these inclusions have any prognostic value. In three recent studies3,5,6 it was indicated that these inclusions can be used to help with diagnosis, but are not prognostic for course of disease. Rodriguez followed a patient with asymptomatic Rai stage 0 CLL. This patient consistently had inclusions noted in lymphocytes for 9 years before any progression of disease was noted.6 In the medical field even if one study reports no prognostic significance, this opinion could change in the future with more studies. Could these crystalline inclusions be used to forecast time to first treatment (TFT) or overall survival?(OS). So far, because of the rarity of these cytoplasmic inclusions, there is no evidence of prognostic value. As well, the mechanism related to their formation and their role in CLL is yet to be determined.

Our case study patient and the various reports found in literature had common flow cytometry immunophenotypes. Patients were all either previously diagnosed with CLL or lymphocytic lymphoma, or were diagnosed at the time of the findings of these inclusions. While these crystalline inclusions alone are not considered diagnostic for CLL, their recognition can be used to assist in a prompt diagnosis of a lymphoproliferative disease. And they are so pretty! What medical laboratory scientist doesn’t love pretty cells? Be like that mouse. Be curious, keep your eyes open, and be on the lookout for these interesting cells in CLL patients, but, more importantly, in patients with lymphocytosis without a known diagnosis of a lymphoproliferative disorder.

References

  1. AJMC, January 7, 2019
  2. Chronic Lymphocytic Leukemia: An Overview of Diagnosis, Prognosis, and Treatment
  3. Huang, Y., Zhang, L. Intracellular rod-like crystals in chronic lymphocyte leukemia. Int J Hematol 112, 267 (2020). https://doi.org/10.1007/s12185-020-02933-7
  4. Numeroff, Laura If You Give a Mouse a Cookie. 1986
  5. Ramlal, B, DiGiuseppe, JA. Intracytoplasmic crystalline inclusions in chronic lymphocytic leukemia. Clin Case Rep. 2019; 7: 1460– 1461. https://doi.org/10.1002/ccr3.2250
  6. Cecilia M. Rodríguez, Carmen Stanganelli, Claudio Bussi, Daniela Arroyo, Darío Sastre, Viviana Heller, Pablo Iribarren & Irma Slavutsky (2018)Intracytoplasmic filamentous inclusions and IGHV rearrangements in a patient with chronic lymphocytic leukemia, Leukemia & Lymphoma, 59:5,1239-1243, DOI: 10.1080/10428194.2017.1370549

-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.