Microbiology Case Study: A Man with History of ALL Presents with Fever and Diarrhea

Clinical History

A man in his 40’s with a past medical history of acute lymphoblastic leukemia/lymphoma (in remission), multiple infections including bacteremia and pulmonary aspergillosis, presented to the hospital with fever and diarrhea. Over the course of his stay, he had worsening renal function and developed profound hypotension and shock, which prompted initiation of two vasopressors and high-dose steroids. Eventually he developed acute hypoxic respiratory failure, requiring intubation. Complete blood count demonstrated an absolute eosinophilia of 8.58 x109/L (reference range 0.04-0.62 x109/L). Imaging revealed bilateral pulmonary infiltrates and a pleural effusion. Respiratory culture with gram stain was ordered for his tracheal aspirate, which revealed few polymorphonuclear cells, many gram-negative rods, yeast, and larvae of Strongyloides stercoralis (Image 1A). Wet mounts of the tracheal aspirate revealed numerous larvae and a few eggs of S. stercoralis (Image 1B-C); many of the larvae were motile (Movie 1). Stool examination of ova and parasites (O & P) were positive for larvae. Given the burden of organisms and prior administration of steroids, he was diagnosed with severe strongyloidiasis, consistent with hyperinfection. Concurrent blood cultures grew Enterococcus faecalis and Stenotrophomonas maltophilia; the respiratory culture also grew S. maltophilia, and tracks from the migrating larvae were observed on respiratory culture bacterial media (Image 1D).

Image 1. Tracheal aspirate Gram stain with S. stercoralis larvae, 100x objective magnification (A). Wet mount of tracheal aspirate revealing larvae (B) and eggs (C), 40x objective magnification. Blood agar plate growing S. maltophilia in an abnormal pattern, indicating motile larvae tracking through the agar (D).

Discussion

Strongyloidiasis is a spectrum of clinical disease caused by the nematode Strongyloides stercoralis.1,2 Descriptions of acute infection have been described in other Lablogatory entries here,3,4 and the full lifecycle is described in detail on the CDC DPDx website.5

Severe strongyloidiasis includes the syndromes of hyperinfection and disseminated disease. Hyperinfection is when there is an elevated burden of the typical autoinfection cycle involving the lungs and GI-tract. Usually there is an antecedent immunosuppressive event, such as administration of corticosteroids. Within the GI-tract lumen, increased numbers of rhabditiform larvae transform into the infective filariform larvae, which traverse the GI mucosa, migrate to the lungs via bloodstream/lymphatics where they enter alveolar air spaces, then ascend the respiratory tract, and are coughed up by the host and swallowed to re-enter the GI tract. In the GI tract adult females lay eggs through parthenogenesis, which give rise to further rhabditiform larvae. In extreme cases of hyperinfection, adults can be found in the lungs, where they may also lay eggs. Finding eggs in respiratory specimens is unusual, and may be related to the burden of disease.6

Disseminated disease is when larvae can be found in any additional organs/organ systems, such as the central nervous system, kidneys, liver, adrenals, etc. Invasive sampling is not typically performed, and larvae can be observed at autopsy.

Laboratory diagnosis of S. stercoralis involves identification of rhabditiform larvae in stool O &P exam; the presence of adults or eggs in stool is rare. Rhabditiform larvae have short buccal cavities and an ovoid genital primordium structure midway through the body (Movie 2). O&P exams can be performed on other body fluids, such as sputum and CSF. Serology can be useful to identify past exposure, especially prior to initiating immunosuppressive therapeutics such as corticosteroids. A nonspecific finding can be observed, as in this case, in the complete blood cell count and differential. Relative and absolute eosinophilia can be found in patients with parasitic infections; therefore, it is reasonable to rule out parasitic infection in this subset of patients. In the case presented here, the absolute eosinophilia was likely due to a persistent S. stercoralis infection, since these nematodes can live in the human host for decades.

The treatment of choice for severe strongyloidiasis is oral ivermectin, though albendazole is an alternative therapy. In some instances, subcutaneous ivermectin administration may be used.7

Follow-up

Oral ivermectin was administered to treat the strongyloidiasis and antibiotics were administered to treat the bacterial infections. Over the coming days, serial tracheal aspirates continued to reveal many larvae and eggs, so therapy was escalated to subcutaneous ivermectin. Over the course of therapy, the patient developed a fungemia with Candida guilliermondii. Despite aggressive antimicrobial therapy and intensive care, the patient remained hypoxemic and hypotensive. The family decided to transition to comfort measures and the patient passed away.

References

  1. Maguire JH. Intestinal Nematodes (Roundworms), in Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases, B. Mandell, Dolin, Editor. 2010, Elsevier: Philadelphia, PA. p. 3577-3586.
  2. Parasitology, in Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, Procop et al., Editors. 2017, Lippincott Williams & Wilkins: China. p. 1452-1454.
  3. Kaur J, Stempak L. An 81 Year Old Female with Persistent Fevers. Lablogatory 2019 [cited 2019 11/5/2019]; Available from: https://labmedicineblog.com/2019/04/23/microbiology-case-study-an-81-year-old-female-with-persistent-fevers/.
  4. Mohammed M, Wojewoda C. A 47 Year Old Male with Abdominal Pain and Diarrhea. Lablogatory 2016 [cited 2019 11/5/2019]; Available from: https://labmedicineblog.com/2016/05/16/microbiology-case-study-a-47-year-old-male-with-abdominal-pain-and-diarrhea/.
  5. Centers for Disease Control. Strongyloidiasis. DPDx 2019 [cited 2019 11/5/2019]; Available from: https://www.cdc.gov/dpdx/strongyloidiasis/index.html.
  6. Keiser PB and Nutman TB. Strongyloides stercoralis in the Immunocompromised Population. Clin Microbiol Rev, 2004. 17(1): p. 208-17.
  7. Hurlimann E and Keiser J, A single dose of ivermectin is sufficient for strongyloidiasis. Lancet Infect Dis, 2019. 19(11): p. 1150-1151.

-IJ Frame, MD, PhD, Microbiology Fellow, University of Texas Southwestern Dallas, Texas

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

The Impact of Fun

“Never, ever underestimate the importance of having fun,” said Randy Pausch, a professor of computer science at Carnegie Mellon University. Indeed, having fun is an important component of life, and that includes your professional life. However, having fun in the workplace can seem like an impossible task sometimes. There is, after all, lots of work to be accomplished, performance to be measured, and projects to complete. This can make it challenging to find of time and ways to have fun appropriately and constructively.

The benefits of having fun in the workplace are plentiful. Because most fun activities require people to work in groups or teams, the shared experience can increase collaboration, engagement, and foster communication. Having fun fosters motivation and commitment to an organization as people associate the positive feelings and experiences with the workplace. This also increases morale and comradery among the participants, which increases their performance. All these aspects, in turn, foster creativity, innovative thinking, and problem-solving skills. The more creative employees are, the more comradery they feel among themselves, and the better they perform the more turnover is reduced. Having fun in the workplace is incredibly beneficial to both the employees and the organization overall.

In today’s workplace culture, people are generally more aware and considerate of what is appropriate behavior. This also applies to having fun, because if activities are only fun and enjoyable if they are appropriate for everyone involved. It is, therefore, important to establish clear boundaries: what is considered part of this activity and what is not. It is also important to consider different levels of physical, mental, and emotional ability. Having fun is inclusive and collaborative, so it is critical to design activities that everyone can partake in. The activity should also always be optional. Making participation mandatory is not actually fun for people, so make sure that there is an opt-in and opt-out option. Finally, every activity should have some element of learning and education. If you are asking people to participate in a fun activity, ensure that they are learning something about one another or about a specific topic.

There are many different ways in which you can incorporate fun in the workplace. Last year at ASCP, our social committee hosted an ‘Oscar Party” in which we could vote for our colleagues in categories such as “Outside the Box Thinker/Innovator,”, “Outstanding Philanthropist,” and “Rookie of the Year.” Then all staff gathered in the kitchen area of our office that was decorated with a red carpet and we all received a glass of sparkling cider. The winners were announced and cheered on as they walked the red carpet. They gave a short speech after receiving their little Oscar award. It was a simple way to have some collective fun and it felt so great cheering everyone on and recognizing certain employees for their outstanding contribution to the society.

On average, babies laugh about 400 times a day. Adults, on the other hand, only laugh about 35 times a day and significantly less often on weekdays than on weekend (Beard, 2014). Laughter is incredibly important to our overall well-being and performance. In fact, “laughter relieves stress and boredom, boosts engagement and well-being, and spurs not only creativity and collaboration but also analytic precision and productivity”(Heggie, 2018). So, let’s try to incorporate more fun and more laughter in both our personal and our professional lives. Let’s find ways to cheer each other up and create a collaborative, warm, and productive environment that fosters engagement, retention, and analytic precision. After all, laughter is the best medicine.

-Lotte Mulder, EdM, is the Senior Manager of Organizational Leadership and Patient Engagement at ASCP. She earned her Masters of Education from the Harvard Graduate School of Education in 2013, where she focused on Leadership and Group Development. After she graduated, Lotte started her own consulting company focused on establishing leadership practices in organizations, creating effective organizational structures, and interpersonal coaching. She has worked in Africa, Latin America, Asia, and the U.S. on increasing leadership skills in young adults through cultural immersion, service learning and refugee issues, and cross-cultural interpretation. She is currently working toward a PhD in Organizational Leadership.


During the 2019 ASCP Annual Meeting in Phoenix, I noticed a morning workshop session entitled “The Impact of Fun.” The title intrigued me, so decided to take a break from the science and clinical medicine workshops that I would normally attend, and take advantage of the opportunity to listen in. 

I have been working as a pathologist and lab director for 30 years, and while I hate to admit it, I had never thought seriously about taking time during the day for playing games with my co-workers. I was always consumed with meetings, deadlines, and getting the clinical work completed.

At the beginning of the course, I was a little unsure what I had gotten myself into. However by the time the workshop concluded, the reality of what I had been missing had set in.

When I returned to work following the meeting, I began to search for fun activities that our lab team could do over a lunch hour. I set a date and promised food to entice the wary into attending the event in the conference room. Once they had assembled, I divided the group into two teams by drawing an imaginary line down the middle of the room. We then played team trivia using a book of questions I had acquired. By the end of the hour, everyone was laughing and having fun. The lab continued to buzz with talk and occasional laughter all afternoon.

We have continued setting aside one noon hour each month where we gather for different types of games. Charades, and Pictionary have been hits. Mostly everyone brings their own lunch, but food or deserts are provided on occasion to keep these events special. There are a few who choose not to participate, but even they occasionally show up to watch and laugh along with the rest. As is pointed out above, you cannot make having fun a mandatory or it ceases to be fun.

Our lab staff really seem to enjoy these events and so does this old pathologist. During our most recent event, one of my young colleagues remarked how much fun these lunches have been, and that they hoped we would continue these going forward. I intend to keep these going as long as I continue working. It has provided me with an opportunity to get to know each of my co-workers much better. I only wish I had learned about the importance of having fun with your co-workers and teammates earlier in my career. I encourage other pathologists, lab directors and section supervisors to learn from my experience and begin finding ways to bring the fun back into the workplace if you have not already done so. 

-Dr. Wisecarver is currently Professor Emeritus in the Department of Pathology/Microbiology at the University of Nebraska Medical Center in Omaha, Nebraska.  He served as Medical Director of the Clinical Laboratories for Nebraska Medicine, their clinical affiliate from 1996 until 2017.  He currently serves as the Director of the Histocompatibility Laboratory for Nebraska Medicine.

Microbiology Case Study: 77 Year Old Man With History of Travel to India

Case Report

A 77 year old male presented to the hospital with chest pain, lightheadedness, burning urination for the past few weeks. He has blood in his urine due to a previously diagnosed neoplasm. The patient moved from India to the United States in February, with a diagnosis of bladder cancer and a history of hypertension, congestive heart failure, coronary artery disease, and atrial fibrillation. In the hospital, abscesses on both right and left kidneys were found, and patient had nephrostomy tubes placed. Purulent discharge confirmed he had a severe urinary tract infection.

Laboratory Identification

The patient’s urine culture grew >100,000 colony forming units/milliliter (CFU/ml) of an oxidase-positive, non-lactose fermenting Gram-negative rod. On the blood agar plate, large gray, smooth, flat, mucoid, β-hemolytic colonies were found. Although bacteria growing on solid media should not be actively smelled, the organism emitted a grape or tortilla smell from the plate. The organism was identified as Pseudomonas aeruginosa by MALDI-TOF mass spectrometry. The isolate was plated onto Mueller Hinton agar for Kirby-Bauer disc diffusion antibiotic susceptibility testing (Image 1). A fluorescent green lawn of bacteria grew up to the edge of all discs, indicating high-level resistance to all antibiotics tested (Table 1). Modified carbapenem inactivation method (mCIM) testing was positive and Cepheid GeneXpert CarbaR PCR testing revealed that this P. aeruginosa isolate carried the New Delhi metallo-β-lactamase-1 carbapenemase (NDM-1).

Image 1. Kirby-Bauer disc diffusion was used for antimicrobial susceptibility testing. Note no zones around any of the antibiotic discs, indicating resistance to all antimicrobials tested.
Table 1. Antimicrobial susceptibility testing interpretations. All drugs tested were resistant to this P. aeruginosa isolate.

Discussion

The issue of super bugs is on the rise, with the fear of antibiotic resistance disseminating through more bacterial populations and species. Carbapenems are drugs that are very powerful broad-spectrum antibiotics, usually reserved as a last resort treatment for serious and resistant infections.1 β-lactamases are divided into four Ambler classes: A, B, C, and D. Class B differs from the others because it utilizes zinc as a metal cofactor for its catalytic activity. The others use a serine residue for their catalytic activity.2

NDM-1 is a class B β-lactamase. It was named after New Delhi, India when a Swedish resident presented with an extremely resistant infection after a trip to India in 2008. NDM-1 bacteria can now be found with high prevalence in India and China, and increasingly in other countries such as the UK and US.3,4 While the origination of the gene may not have been India, many of these infections are from people who have traveled to India or other Asian continents.5 Concerns about overprescribing and misuse of antibiotics in India are rising, where India is one of the biggest consumers of antibiotics in the world. One study even found striking evidence of this misuse, demonstrating that 2 out of 3 adults under 20 presented antibiotic resistance isolates to fluoroquinolones and/or cephalosporins.6,7,8

Image depicting the NDM-1 protein anchored in the outer membrane of the bacterium. (Taken from Bahr, Guillermo, et al. “Clinical Evolution of New Delhi Metallo-β-Lactamase (NDM) Optimizes Resistance under Zn(II) Deprivation.” Antimicrobial Agents and Chemotherapy, vol. 62, no. 1, 2017, doi:10.1128/aac.01849-17.)

The gene for NDM-1 is blaNDM-1 and has been found on both plasmid and chromosomal components of different bacteria. Due to its presence on plasmids, the gene can easily spread through bacterial populations and other bacterial species – as has already been documented in Enterobacteriaceae and A. baumannii.3 The β-lactamase that it codes for is a lipoprotein that is anchored in the outer membrane of the gram negative bacteria. Other metalo-β-lactamases (MBLs) are periplasmic proteins, which are more affected by changes in essential metal cofactors in their enzymatic function. Thus far, it has been found that there are 16 discovered variants of NDM. Some variants being more fit than NDM-1. It is hypothesized that these variants are being selected for in the clinical setting, with the protein being more stable and demonstrating higher affinities for zinc during time of metal-chelating (a process the immune system adapts to combat infections).9 Unfortunately, NDM-1 and its variants are resistant to almost all antibiotics. Usually the only option is colistin and tigecycline.3

The disturbing issue, and the big picture, is the capability of MDR organisms and their genes of disseminating. As previously mentioned, NDM-1 is capable of spreading to other species and within its population. Yet, a terrifying report has demonstrated blaNDM-1 detection in artic soil samples from 2013, 4 years after the first detection of the gene.10 This demonstrates the ability for antibiotic resistance to spread on a global scale, and how serious this battle truly is.

References

  1. “Carbapenem-Resistant Enterobacteriaceae (CRE) Infection.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 23 Feb. 2015, http://www.cdc.gov/hai/organisms/cre/cre-patientfaq.html.
  2. Walther-Rasmussen, Jan, and Niels Høiby. “Class A Carbapenemases.” Journal of Antimicrobial Chemotherapy, vol. 60, no. 3, 2007, pp. 470–482., doi:10.1093/jac/dkm226.
  3. Khan, Asad U., et al. “Structure, Genetics and Worldwide Spread of New Delhi Metallo-β-Lactamase (NDM): a Threat to Public Health.” BMC Microbiology, vol. 17, no. 1, 2017, doi:10.1186/s12866-017-1012-8.
  4. Mohapatra P. R. (2013). Metallo-β-lactamase 1–why blame New Delhi & India?. The Indian journal of medical research137(1), 213–215.
  5. Roos, Robert. “Canada Finds More Infections with NDM-1 Resistance Factor.” University of Minnesota, Center for Infectious Disease Research and Policy, 11 Nov. 2010, http://www.cidrap.umn.edu/news-perspective/2010/11/canada-finds-more-infections-ndm-1-resistance-factor.
  6. Gupta, M., Didwal, G., Bansal, S., Kaushal, K., Batra, N., Gautam, V., & Ray, P. (2019). Antibiotic-resistant Enterobacteriaceae in healthy gut flora: A report from north Indian semiurban community. The Indian journal of medical research, 149(2), 276–280. doi:10.4103/ijmr.IJMR_207_18
  7. Kotwani, Anita, and Kathleen Holloway. “Access to Antibiotics in New Delhi, India: Implications for Antibiotic Policy.” Journal of Pharmaceutical Policy and Practice, vol. 6, no. 1, 2013, doi:10.1186/2052-3211-6-6.
  8. Kotwani, Anita, et al. “Antibiotic-Prescribing Practices of Primary Care Prescribers for Acute Diarrhea in New Delhi, India.” Value in Health, vol. 15, no. 1, 2012, doi:10.1016/j.jval.2011.11.008.
  9. Bahr, Guillermo, et al. “Clinical Evolution of New Delhi Metallo-β-Lactamase (NDM) Optimizes Resistance under Zn(II) Deprivation.” Antimicrobial Agents and Chemotherapy, vol. 62, no. 1, 2017, doi:10.1128/aac.01849-17.
  10. Mccann, Clare M., et al. “Understanding Drivers of Antibiotic Resistance Genes in High Arctic Soil Ecosystems.” Environment International, vol. 125, 2019, pp. 497–504., doi:10.1016/j.envint.2019.01.034.

-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. He graduated with a bachelors in Biology at the University of Illinois at Chicago (UIC) and concurrently from the UIC Honors College. He discovered his passion for the lab through his experience in healthcare. His interests include microbiology, molecular, immunology, and blood bank.

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

A 66 Year Old Male with Diarrhea, Weight Loss, and Night Sweats

Case History

A 66 year old man with past medical history of recently diagnosed Clostridioides difficile colitis presented to emergency department with diarrhea, weight loss of 52 pounds in 4 months, and occasional night sweats. CT imaging revealed dilation of small bowel with thickened mucosal folds. The duodenum was subsequently biopsied to reveal diffuse intestinal lymphangiectasia containing PAS positive and Congo red negative eosinophilic material and lamina propria foamy macrophages. Laboratory investigations revealed normocytic anemia, proteinuria, and peripheral IgM kappa monoclonal gammopathy.

Biopsy Findings

Image 1. Aspirate.
Image 2. Core biopsy.
Image 3. CD138.
Image 4. Kappa ISH.
Image 5. Lambda ISH.

Bone marrow aspirate shows increased plasma cells and mast cells. H&E stained sections demonstrate a normocellular bone marrow with trilineage hematopoiesis and involvement by 35% plasma cells. By immunohistochemistry, CD138 highlights clusters of plasma cells that predominantly express kappa light chain restriction.

FISH and Mutation Analysis

FISH demonstrated loss of chromosome 11 and gain of chromosome 15, which was consistent with plasma cell dyscrasia. MYD88 mutation analysis did not detect the mutation.

Diagnosis

The findings of the patient’s normocytic anemia, IgM monoclonal gammopathy, and intestinal lymphangectasia with an associated plasma cell dyscrasia involving the bone marrow favor a lymphoplasmacytic lymphoma/Waldenström macroglobulinemia.

Discussion

Waldenstrom macroglobulinemia (WM) is a malignant B-cell lymphoproliferative disorder characterized by lymphoplasmacytic infiltration of the bone marrow and peripheral IgM monoclonal gammopathy.1 It is rare with an overall incidence of 3 per million persons per year, accounting for 1-2% of hematologic cancers.1 It occurs predominantly in Caucasian males, with a median age of 63-68 years old at diagnosis.1-3

Patient may be asymptomatic for years and require observation or experience a broad spectrum of signs and symptoms. These symptoms may be attributable to the tumor infiltration of the bone marrow and lymphoid tissues, IgM circulating in the blood, and IgM depositing into tissues. The most common clinical presentation of WM is fatigue and nonspecific constitutional symptoms, such as fever, night sweats, and weight loss, due to normochromic, normocytic anemia. 20-30% of patients may exhibit lymphadenopathy and hepatosplenomegaly due to infiltration of peripheral tissues. High concentration of IgM in the circulation may lead to hyperviscosity, resulting in oronasal bleeding, gingival bleeding, blurred vision due to retinal hemorrhages, and neurological symptoms, including headache, ataxia, light-headedness, dizziness, and rarely, stroke.2-3 The gastrointestinal manifestations are rare; however, IgM monoclonal protein may deposit into the lamina propria of the GI tract, causing diarrhea, steatorrhea, and GI bleeding.4 Other IgM-related manifestations include cold agglutinin hemolytic anemia, cryoglobulin, and amyloid deposition in tissues.3

Diagnosis of WM includes evidence of IgM monoclonal gammopathy and at least 10% of bone marrow infiltration by lymphoplasmacytic cells.5 Monoclonal gammopathy can be detected by the monoclonal spike, or M-spike, on serum protein electrophoresis.3 Serum immunofixation may be performed to identify the type of monoclonal protein and the type of light chain involved.3 In terms of immunophenotype, neoplastic cells express surface IgM, cytoplasmic Igs, CD38, CD79a, and pan B-cell markers (CD19, CD20, and CD22). CD10 and CD23 are absent. Expression of CD5 occurs in approximately 5-20% of cases.6 Recent studies have reported two most common somatic mutations in WM, which are MYD88 L265P mutations (90-95% of cases) and CXCR4 (30–40% of cases).7 Absence of these mutations, however, do not completely exclude the diagnosis of WM.

The International Staging System for WM identifies five factors associated with adverse prognosis, including age older than 65, hemoglobin < 11.5g/dL, platelet count < 100K/μL, beta-2-microglobulin > 3mg/L, and monoclonal IgM concentration > 7g/L.3 Patients younger than the age of 65 years with 0 or 1 of these factors are in the low-risk category with a median survival of 12 years.3 In contrast, patients with 2 or more risk factors are in the intermediate- and high-risk categories and have a median survival of almost 4 years. 3

Management of WM depends on the patient’s clinical manifestations.Furthermore, patients with minimal symptoms should be managed with rituximab, whereas patients with severe symptoms related to WM should receive more aggressive treatment, including dexamethasone, rituximab and cyclophosphamide. Hyperviscosity syndrome is an oncologic emergency that requires removal of excess IgM from the circulation via plasmapheresis.8

References

  1. Neparidze N, Dhodapkar MV. Waldenstrom’s Macroglobulinemia: Recent advances in biology and therapy. Clin Adv Hematol Onco. 2009 Oct;7(10): 677-690.
  2. Leleu X, Roccaro AM, Moreau AS, Dupire S, Robu D, et al. Waldenstrom Macroglobulinemia. Cancer Lett. 2008 Oct;270(1):095-107.
  3. Tran T. Waldenstrom’s macroglobulinemia: a review of laboratory findings and clinical aspects. Laboratory Medicine. 2013 May;44(2):e19-e21.
  4. Kantamaneni V, Gurram K, Khehra R, Koneru G, Kulkarni A. Distal illeal ulcers as gastrointestinal manifestation of Waldenstrom Macroglbulinemia. 2019 Apr; 6(4):pe00058.
  5. Grunenberg A, Buske C. Monoclonal IgM gammopathy and Waldenstrom’s macroglobulinemia. Dtsch Arztebl Int. 2017 Nov;114(44):745-751.
  6. Bhawna S, Butola KS, Kumar Y. A diagnostic dilemma: Waldenstrom’s macroglobulinemia/plasma cell leukemia. Case Rep Pathol. 2012;2012:271407.
  7. Varettoni M, Zibellini S, Defrancesco I, Ferretti VV, Rizzo E, et all. Pattern of somatic mutations in patients with Waldenstrom macroglobulinemia or IgM monoclonal gammopathy of undetermined significance.
  8. Oza A, Rajkumar SV. Waldenstrom macroglobulinemia: prognosis and management. Blood Cancer Journal. 2015;5:e394.

-Jasmine Saleh, MD MPH is a pathology resident at Loyola University Medical Center with an interest in dermatopathology and hematopathology. Follow Dr. Saleh on Twitter @JasmineSaleh.

–Kamran M. Mirza, MD, PhD, MLS(ASCP)CM is an Assistant Professor of Pathology and Laboratory Medicine, Medical Education and Applied Health Sciences at Loyola University Chicago Stritch School of Medicine and Parkinson School for Health Sciences and Public Health. A past top 5 honoree in ASCP’s Forty Under 40, Dr. Mirza was named to The Pathologist’s Power List of 2018 and placed #5 in the #PathPower List 2019. Follow him on twitter @kmirza.

Microbiology Case Study: A 50 Year Old Male with Fevers

Case History

The patient is a 50 year old male who presented to urgent care with 5 days of fevers, chills, and myalgias. He reports no known tick bites, or prior treatment for tickborne illness, he travels frequently for work and has been in Pittsburgh, Omaha, Philadelphia, Charlotte, and Long Island over the past 3 months and is frequently outside golfing in Vermont and while traveling. At urgent care he had a CBC, CMP and urinalysis. His CBC was remarkable for leukopenia with absolute neutropenia (WBC count 2,790; Absolute neutrophil count 390) and thrombocytopenia (platelet count 26,000/cmm). His CMP was remarkable for a mildly elevated AST (66U/L) and total bilirubin (2.4mg/dL). A peripheral blood smear was made which revealed ring forms in his red blood cells. BINAX testing for malaria was negative. The next day he presented to the emergency department with left upper quadrant abdominal pain, night sweats, fatigue, fever, and blood in his urine where is was informed of his CBC results and was immediately started on azithromycin and atovaquone. Given the severity of his presentation a co-infection with Anaplasma phagocytophilum (Anaplasmosis) was suspected though the PCR testing was negative.

Laboratory Identification

Image 1. Giemsa stain of thin blood smears showing intracellular (left) and extracellular (right) ring-form organisms.

Thick and thin blood smears (recommended) were prepared which showed both intra and extracellular organisms with in normal sized red blood cells. BINAX testing for malaria was negative. Given the appearance and presence both within and outside the cell a diagnosis of Babesiosis was made.

Discussion

Babesiosis in the United States is caused by the microscopic parasite Babesia microti. Occasional cases caused by other species of Babesia have been detected (1). Babesia microti is spread by Ixodes scapularis ticks (also called blacklegged ticks or deer ticks) (1,2). Transmission mainly occurs in parts of the Northeast and upper Midwest; and it usually peaks during the warm months (1). Because Babesia shares a vector and geographic distribution with Borrelia burgdorferi (Lyme disease) coinfection of ticks with these two organisms can be seen in up to 40% of ticks and though the incidence of Babesiosis is much lower; up to 10% of Connecticut patients with seropositivity for Lyme disease are also seropositive for Babesia (2).

Most infections are probably asymptomatic. Manifestations of disease include fever, chills, sweating, myalgias, fatigue, hepatosplenomegaly, and hemolytic anemia. Symptoms typically occur after an incubation period of 1 to 4 weeks, and can last several weeks. The disease is more severe in patients who are immunosuppressed, splenectomized, and/or elderly (1,3).

During a blood meal, a Babesia-infected tick introduces sporozoites into the human host (1,3). Sporozoites enter erythrocytes and undergo asexual replication (budding). Multiplication of the blood stage parasites is responsible for the clinical manifestations of the disease (1). Humans are dead-end hosts and there is probably little, if any, subsequent transmission that occurs from ticks feeding on infected persons (1). However, human to human transmission is well recognized to occur through blood transfusions (1). Diagnosis is often rendered by direct visualization on thick and thin blood smears. Because the percent parasitemia is often low, the organisms can be easily missed, especially by automated hematology analyzers (3). Molecular methods such as PCR can be performed (3), but are not recommended as the first line test as the blood parasite exam with thick and thin blood smears are clinically sensitive in patients with symptomatic disease.

Most mild cases of Babesiosis will resolve spontaneously without treatment, especially in patient with a spleen (1,3). Treatment for more severe disease includes either azithromycin and atovaquone or clindamycin and quinine (1,3). If patients are severely immunocompromised and/or splenectomized can be treated with exchange transfusion in addition to antimicrobials (3).

References

  1. Centers of Disease Control and Prevention: Babesiosis. https://www.cdc.gov/parasites/babesiosis/
  2. Procop, Gary W., et al. Konemans Color Atlas and Textbook of Diagnostic Microbiology. 7th ed., Wolters Kluwer Health, 2017.
  3. Tille, Patricia M. Bailey & Scotts Diagnostic Microbiology. 13th ed., Elsevier, 2014.

-Casey Rankins, DO, is a 3rd year Anatomic and Clinical Pathology resident at the University of Vermont Medical Center.

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

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.