Hematology Case Study: CBC with >80% Blasts

The patient is a 67 year old male who first visited his dentist at the end of December complaining of pain in the jaw that he had been experiencing since early Dec. He had put off making an appointment because he didn’t want to have to go to the doctor with COVID precautions, but the pain was now radiating to his teeth, so he made a dentist appointment. The dentist found no evidence of abscess or other infection but ‘adjusted his bite’. The patient was advised to take over the counter NSAIDs as needed or pain but no prescriptions was needed. Three weeks later the patient visited an urgent care because he had no improvement of the jaw pain. At this time he relayed symptoms of cough, fever, chills, night sweats and chronic fatigue. Patient history included an active lifestyle with vigorous aerobic exercise several times a week, but the he stated that he had been feeling too fatigued to exercise for over a month. On exam the patient was found to be tachycardic with bilateral tonsillar lymphadenopathy and oropharyngeal exudate. The patient was tested for COVID, influenza and Group A Strep. The COVID-19 was negative, as was the influenza A and B, but the Group A Strep was positive. The patient was sent home with a prescription for antibiotics.

One week later, the patient called his PCP because he still had cough, fever and chills and now was experiencing shortness of breath. The office directed the patient to go to the ER but the patient was reluctant to go to the hospital and stated he would rather be seen at the office. On review of the patients chart, the PCP agreed to see him in the office because he had had a negative COVID test in the past week. Two days later the doctor examined the patient in his office and still suspected COVID-19. He ordered a PCR COVID-19 test along with CBC/differential and erythrocyte sedimentation rate (ESR). We received a routine CBC on the patient. Results are shown below.

The patient had no previous hematology or oncology history and no previous CBC received at our lab. The critical WBC was called to the physician. Based on the WBC and flags on the auto differential, a slide was made and sent to our CellaVision (CV). On opening the slide in CV, we immediately called our pathologist for a pathology review. A rare neutrophil was seen on the peripheral smear, with immature appearing monocytes, few lymphocytes and many blasts.

Image 1. Images from CellaVision.

The pathologist reviewed the slide and the sample was sent for flow cytology studies and FISH. The pathologist’s comment ”Numerous blasts (>60%) consistent with Acute Myeloid Leukemia(AML). Specimen to be submitted for flow cytometry. Hematology consult recommended” was added to the report.

Image 2. Image from CellaVision. Predominately blasts with one neutrophil seen in field of unremarkable RBCs.
Image 3. Image from CellaVision.

The myeloid/lymphoid disorders and acute leukemia analysis by flow cytometry reported myeloblasts positive for CD117,CD33, and CD13. Final interpretation was Acute Myeloid leukemia (non-M3 type).

AML is the most common form of leukemia found in adults. AML was traditionally classified into subtypes M0 through M7, based on the cell line and maturity of the cells. This was determined by how the cells looked under the microscope after a series of special staining techniques, but did not take into account prognosis. It is now known that the subtype of AML is important in helping to determine a patient’s prognosis. In 2016 World Health Organization (WHO) updated the classification system to better address prognostic factors. They divided AML into several broad groups, including AML with certain chromosomal translocations, AML related to previous cancer or cancer therapy, AML with involvement of more than one cell type, and other AML that don’t fall into the first three groups.2 Once a case has been placed in one of these broad groups, the AML can be further classified as poor risk, intermediate risk and better risk based on other test results. Better risk is associated with better response to treatments and longer survival.3 The European LeukemiaNET (ELN) first recommended integrating molecular and cytogenic data into classification to create such a risk classification system for AML in 2010 (ELN-2010). In 2017, this was again revised (ELN-2017) to further improve risk stratification. The ELN-2017 can be used to more accurately predict prognosis in newly diagnosed AML.1

What this means is that AML is now classified by abnormal cell type as well as by the cytogenetic, or chromosome, changes found in the leukemia cells. Certain chromosomal changes can be matched with the morphology of the abnormal cells. These chromosomal changes can help doctors determine the best treatment options for patients because these changes can predict how well treatment will work.

Examples of risk classification include the knowledge that some chromosome rearrangements actually offer a better prognosis. For example, a translocation between chromosomes 15 and 17 [t(15;17)] is associated with acute promyelocytic leukemia (APL or M3). APL is treated differently than other subtypes and has the best prognosis of all the AML subtypes. Other favorable chromosomal changes include [t(8;21)] and [inversion (16) or translocation t(16;16)]. Examples of intermediate risk prognosis are ones associated with normal chromosomes and [t(9;11)]. Poor prognosis is associated with findings such as deletions or extra copies of certain chromosomes or complex changes in many chromosomes.3

The patient was diagnosed with AML, non M3 type. AML prognosis is based on CBC results, markers on the leukemia cells (flow cytometry), chromosome (cytogenic) abnormalities found and gene mutations (molecular abnormalities). In this patient the FISH studies did not demonstrate any chromosome rearrangements, which alone would place him in an intermediate risk group. In addition, our patient was over age 60 and had a WBC over 100,000/mm3 which have both been linked to worse outcomes.

Here’s one more photo for your enjoyment! It’s not often that we see so many blasts in a patient with no previous history. As a side note, I was contemplating titling this blog “Fatigue and Shortness of Breath in the Time of COVID.” I can’t help but wonder if this patient would have been diagnosed 6-8 weeks earlier if this was another year and he had been seen when he first experienced symptoms. This year, emergency rooms and physicians have reported a decrease in numbers of patients being seen for chest pain, ketoacidosis, shortness of breath, strokes and other serious conditions. Many patients are reluctant or afraid of sitting in crowded waiting rooms, fearful they will catch COVID. And many doctors are only offering virtual visits or have reduced the number of patients being seen so it is harder to get appointments. This patient expressed his reluctance to seek medical help because of fears of COVID. He did not want to go out in public and waited almost a month for symptoms to go away on their own before first being seen. After going to the walk in center, he called his PCP a week later and was still averse to going to the ER as suggested by the doctor. Then he waited another 2 days for an office appointment. The doctor still suspected COVID, but fortunately for the patient, ordered a CBC. The flow cytometry and FISH studies were available the following day. The patient was referred for hematology consult but has not been seen again at our hospital.

Image 4. More images from CellaVision.

References

  1. Boddu, P.C., Kadia, T.M., Garcia‐Manero, G., Cortes, J., Alfayez, M., Borthakur, G., Konopleva, M., Jabbour, E.J., Daver, N.G., DiNardo, C.D., Naqvi, K., Yilmaz, M., Short, N.J., Pierce, S., Kantarjian, H.M. and Ravandi, F. (2019), Validation of the 2017 European LeukemiaNet classification for acute myeloid leukemia with NPM1 and FLT3‐internal tandem duplication genotypes. Cancer, 125: 1091-1100. https://doi.org/10.1002/cncr.31885
  2. Mandel, Ananya. Acute Myeloid Leukemia Classification. Medical Life Sciences. https://www.news-medical.net/health/Acute-Myeloid-Leukemia-Classification.aspx
  3. Ari VanderWalde, MD, MPH, MA, FACP; Chief Editor: Karl S Roth, MD. Genetics of Acute Myeloid Leukemia. Medscape. Updated: Dec 17, 2018 
Socha-small

-Becky Socha, MS, MLS(ASCP)CMBBCM 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 40 years and has taught as an adjunct faculty member at Merrimack College, UMass Lowell and Stevenson University for over 20 years.  She has worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. She currently works at Mercy Medical Center in Baltimore, Md. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

Case Study: Newborn with Thrombocytopenia and Bruising

A newborn, healthy, full term, male child, was born with bruising on his left thigh and developed petechiae and purpuric hemorrhages several hours after birth. The baby was moved to the NICU for observation and a CBC was ordered by the NICU provider.

  • WBC, RBC, Hgb, Hct and indicies were normal
  • Platelet count 58 x103/μL
  • Baby exhibited no symptoms of sepsis
  • Smear reviewed with no platelet clumping observed

The mother is a 28 year old, gravida 1, para 1 with normal CBC and platelet count. Her prenatal history was unremarkable. She has no history of immune thrombocytopenia (ITP) and no history of being prescribed drugs known to be associated with drug induced thrombocytopenia

Thrombocytopenia is not an uncommon finding in neonates, particularly in the neonatal intensive care unit (NICU). In preterm infants, the most common causes of thrombocytopenia are complications of pregnancy, including pregnancy-induced hypertension (PIH), intrauterine growth retardation, preeclampsia ,and HELLP syndrome (hemolytic anemia, elevated liver enzymes, low platelet count). Examination of a peripheral smear in these patients will typically reveal neutropenia with densely packed red cells, increased nucleated RBCs and deceased platelet estimate. These placental insufficiency cases typically occur within the first 72 hours of life, platelet counts are >50 x 103/μL, resolve without treatment and require no further investigation. On the other hand, thrombocytopenia in preterm infants that develops after 72 hours is most likely due to sepsis or necrotizing enterocolitis and requires investigation and treatment.2

In an otherwise healthy appearing full term infant, the most common cause of thrombocytopenia in the first 72 hours of life is neonatal alloimmune thrombocytopenia (NAIT). When a platelet count drops below 150 x 103/L in these newborns, it is important to investigate the thrombocytopenia. The first step is to always check a peripheral smear for clumping to rule out spurious thrombocytopenia. With a low platelet count and the absence of spurious thrombocytopenia, NAIT can be suspected. This condition is similar in pathogenesis to hemolytic disease of the fetus and newborn (HDFN), and is caused by an incompatibility in human platelet antigens between mother and baby. In about 80% of cases, the mother is found to be HPA-1b and the father and baby are HPA-1a.1 The mother forms anti-HPA-1a which crosses the placenta and destroys the fetus’ platelets. Most cases of NAIT are asymptomatic, or cause only mild bleeding, and resolve in 1-2 weeks.1

Although many cases of NAIT are mild, it is important to recognize because it can be a life-threatening disorder. With more severe thrombocytopenia, in both premature and full term infants, NAIT can result in intracranial bleeding either before birth or shortly after birth. NAIT can also cause long term neurologic complications. Therefore, when a neonate is suspected to have NAIT, he should be screened for intracranial hemorrhage. Since mothers are most often found to have anti- HPA-1a, and the second most commonly found antibody is anti-HPA-5b, neonates with platelet counts <30 x 103/L should be transfused with antigen matched or HPA-1a and HPA-5b negative, CMV negative, single donor apheresis platelets.

It is important to note that NAIT can occur in a first pregnancy but subsequent pregnancies are usually more severely affected. In confirming NAIT after a first delivery or monitoring a subsequent pregnancy, serological testing should be done on both parents to determine the risk of having an infant born with NAIT. If the father is homozygous for the antigen which the mother lacks, 100% of infants would be at risk. If the father is heterozygous, an infant would have a 50% chance of inheriting the antigen from the father.

NAIT in a first pregnancy is typically unrecognized until after birth. Some groups have advocated for routine prenatal screening for NAIT in all pregnant women, but this is costly and still debated. It is agreed that after an affected first child, subsequent pregnancies should be monitored closely. In at risk pregnancies, weekly antenatal IVIg infusions should be used during pregnancy to help prevent fetal bleeding.3

The mother in this case was tested and found to be HPA-1a negative with anti-HPA-1a. The father was also tested and found to be HPA-1a positive. The infant’s platelet counts began to increase at 7 days, with no further bleeding. The mother was referred to a NAIT specialty team for future pregnancies.

Diagnosis: Neonatal Immune Thrombocytopenia (NAIT)

  • Similar in pathogenesis to hemolytic disease of the fetus and newborn (HDFN)
  • Incompatibility in human platelet antigens between mother and baby.
  • Can affect first born
  • In majority of cases, the mother is HPA-1b and the father and baby are HPA-1a
  • Second most common is anti-HPA-5b

References

  1. http://naitbabies.org/wp-content/uploads/141208_NAIT_Registry_poster.pdf
  2. Subarna Chakravorty and Irene Roberts. How I manage neonatal thrombocytopenia . Blackwell Publishing Ltd, British Journal of Haematology. 2011; 156, 155–162
  3. T.W. de Vos, D. Winkelhorst, M. de Haas, E. Lopriore, D. Oepkes. Epidemiology and management of fetal and neonatal alloimmune thrombocytopenia. Transfusion and Apheresis Science. 2020
  4. Shamudheen Rafiyath, Immune Thrombocytopenia and Pregnancy Treatment & Management Updated: Sept. 2020 https://emedicine.medscape.com/article/208697-treatment

-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.

Microbiology Case Study: A 40 Year Old Man with LVAD Exit Site Pain

Case history

A 40 year old male with a history of cardiomyopathy requiring a left ventricular assist device (LVAD) was seen in clinic with a complaint of pain at the exit site of the LVAD driveline. History is notable for multiple admissions for driveline-associated complications. Despite extensive prior evaluation, cultures and imaging of the driveline exit site had been repeatedly negative with the exception of a methicillin-susceptible Staphylococcus aureus.This was treated with nafcillin, followed by doxycycline for oral suppression. The patient had stopped taking oral antibiotics two months prior to presentation. Imaging revealed a 1.4 cm region around the driveline exit site suggestive of either phlegmon, hematoma, or a developing abscess. Blood cultures and cultures of the driveline exit site were collected and sent to the clinical microbiology laboratory. Upon physical examination, the driveline exit site was tender, but no erythema was noted. The patient endorsed intermittent rust-colored drainage from the site. Blood cultures remained negative for the duration of the patient’s hospital course, and the patient was discharged on nafcillin with plans to transition to doxycycline.

Laboratory identification

The Gram stain of the driveline exit site was unremarkable, with no organisms and few neutrophils seen. Aerobic cultures yielded a light amount of gram positive cocci in addition to mixed skin flora. Colonies were small, and weakly beta hemolytic on blood agar (Image 1A). This organism was catalase- and coagulase-positive, and definitively identified as Staphylococcus aureus by MALDI-TOF MS. Susceptibility testing was performed by broth microdilution, where the organism was determined to be a vancomycin-intermediate Staphylococcus aureus (VISA, MIC=4, Image 1C). Due to the unusual nature of the result, it repeated and confirmed by E-test (Image IB) in our laboratory, and independently verified at our contract reference laboratory. The isolate was also referred to the Texas State Public Health Laboratory where the vancomycin-intermediate phenotype was again confirmed. This isolate was also daptomycin non-susceptible, but remained susceptible to oxacillin, trimethoprim/sulfamethoxazole, linezolid, rifampin, and clindamycin.

Image 1. A. Growth of the weakly beta-hemolytic vancomycin-intermediate S. aureus strain on blood agar. B. Measurement of vancomycin susceptibility by E-test (MIC=4). C. Confirmation of the VISA phenotype by broth microdilution (MIC=4).

Discussion

All models of LVADs require a percutaneous driveline which is a link between the implanted device and the external power source.1In addition to providing power, the driveline also provides controlling and sensing functions for the LVAD.2 The driveline exit site is one of the most common sites of LVAD infection as the driveline creates a conduit for entry of bacteria from the external environment. Additionally, the prosthetic material of the driveline can serve as an ideal substrate for biofilms formation.1 The most common microorganisms associated with LVAD-related infections members of the skin microbiota (i.e. staphylococci), Pseudomonas sp., and enteric bacteria.3

Staphylococcus aureus remains an important human pathogen globally. While antibiotic intervention remains a mainstay of treatment, the emergence of resistance has historically changed the way patients are managed. Mobile genetic elements (including plasmids and transposons) are important mediators of antibiotic resistance in S. aureus, particularly with respect to beta-lactams and glycopeptide antibiotics. Due to the widespread emergence of beta-lactamase conferred penicillin-resistance, semisynthetic penicillinase-resistant penicillins (including methicillin, oxacillin, and nafcillin) were developed for clinical use in the late 1950s. However, resistance to these compounds in S. aureus was reported only a few years following their introduction. Vancomycin became the antibiotic of choice for methicillin-resistant S. aureus (MRSA) therapy in the 1980s, and contemporary management remains largely reliant on this antibiotic despite the recent availability of newer agents from different antibiotic classes.4Thus, vancomycin non-susceptibility among S. aureus isolates is a rare phenomenon with serious clinical implications, with only modest increases in vancomycin MICs resulting in treatment failures.5

The first vancomycin-intermediate S. aureus (VISA) isolate was reported in 1997 in Japan, followed by the first vancomycin-resistant isolate in 2002 in the US.4 It is important to note that the mechanisms driving these two phenotypes are entirely different. The fully vancomycin-resistant phenotype is due to the acquisition of the vanA gene which confers cell wall alterations that prohibit vancomycin from efficiently binding its target. By contrast, the vancomycin-intermediate phenotype remains less well described mechanistically, but VISA strains share similar phenotypic traits. These include: alterations in growth kinetics, increased cell wall thickness, a reduction in peptidoglycan crosslinking, decreased autolysis, altered surface protein profile, and variation of expression levels of global genetic regulators.4,5 These phenotypes are due to mutations and alterations in expression of a number of candidate genes involved in cell wall synthesis, capsule production, and global regulators of virulence.

The emergence of a VISA phenotype is usually found in the setting of MRSA strains that have been treated with prolonged vancomycin therapy.5 However, in this patient’s case, vancomycin had only been utilized infrequently for unrelated infections several years prior. Daptomycin had not previously been used in this patient’s clinical care. This VISA isolate was also oxacillin-susceptible which is a less common finding among reported VISA strains. While exposure of S. aureus to non-glycopeptide antibiotics including beta-lactams can trigger VISA phenotypes in vitro,6 it is currently not possible to elucidate the mechanism underpinning vancomycin non-susceptibility, nor what has driven this resistant phenotype, in this patient’s isolate. The patient currently is doing well on doxycycline suppressive therapy after completing his course of nafcillin, and continues to be monitored through follow-up appointments.

References

  1. Leuck A-M. 2015. Left ventricular assist device driveline infections: recent advances and future goals. Journal of Thoracic Disease 7:2151-2157.
  2. Long B, Robertson J, Koyfman A, Brady W. 2019. Left ventricular assist devices and their complications: A review for emergency clinicians. The American Journal of Emergency Medicine 37:1562-1570.
  3. Zinoviev R, Lippincott CK, Keller SC, Gilotra NA. 2020. In Full Flow: Left Ventricular Assist Device Infections in the Modern Era. Open Forum Infectious Diseases 7.
  4. McGuinness WA, Malachowa N, DeLeo FR. 2017. Vancomycin Resistance in Staphylococcus aureus
The Yale Journal of Biology and Medicine 90:269-281.
  5. Gardete S, Tomasz A. 2014. Mechanisms of vancomycin resistance in Staphylococcus aureus. The Journal of Clinical Investigation 124:2836-2840.
  6. Roch M, Clair P, Renzoni A, Reverdy M-E, Dauwalder O, Bes M, Martra A, Freydière A-M, Laurent F, Reix P, Dumitrescu O, Vandenesch F. 2014. Exposure of Staphylococcus aureus to subinhibitory concentrations of β-lactam antibiotics induces heterogeneous vancomycin-intermediate Staphylococcus aureus. Antimicrobial agents and chemotherapy 58:5306-5314.

-Zoya Khan MS, MLS(ASCP)CM is a medical technologist in the clinical microbiology laboratory at UT Southwestern with almost 10 years’ experience. She received a BS in Medical Technology from Texas Women’s University, and an MS in Clinical Practice Management from Texas Tech Health Science Center. She has an active interest in mycology and laboratory assay verification.

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

-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.

Microbiology Case Study: A 30 Year Old with Fever Post Stem Cell Transplant

Case History

A thirty year old female with refractory acute leukemia was admitted to undergo allogeneic stem cell transplantation. Her initial admission had multiple infectious complications and chemotherapy-induced pancytopenia with profound absolute neutropenia. The patient was placed on prophylaxis/therapy including bacterial, viral, and fungal coverage. On hospital day 14, the patient was febrile to 38°C; vancomycin/piperacillin-tazobactam were added as empiric therapy due to concern for sepsis with fluctuation in mental status. CT Brain without contrast revealed a large intracranial hematoma with mass effect. Her mental status continued to decline and intubation was required for airway protection. An emergent decompressive hemicraniectomy was performed where necrotic brain tissue with hemorrhage/clot were found.

Due to ongoing fevers, empiric antimicrobial therapy was further broadened with meropenem, doxycycline, trimethoprim/sulfamethoxazole, and liposomal amphotericin B (L-AMB). Repeat cultures and imaging studies were ordered to evaluate for infection as a fever source. CT Angiography Chest (Image 1A-C) was performed and revealed an extensive non-enhancing area of ground glass opacity with peribronchovascular consolidation (“Reversed Halo” sign) in the right lung concerning for angioinvasive fungal infection. A tracheal aspirate was sent for bacterial and fungal culture. Subsequent bronchoscopy revealed extensive necrosis involving all visualized airways of the right tracheobronchial tree to the first subsegmental level (Image 1D). By contrast, the left lung appeared relatively normal and uninvolved. Bronchial washings of the right lung were also submitted for culture.

Image 1. Computed Tomography (CT) Angiography Chest images: Coronal (A), Cross-section (B) and Sagittal (C) sections reveal a large central ground glass opacity surrounded by a dense consolidation in the shape of a ring “Reversed Halo” sign. Bronchoscopy (D) revealed extensive necrosis and friable mucosa in the visible airways of the right lung.

Laboratory Identification

Respiratory specimens were sent to the microbiology laboratory for bacterial and fungal cultures. Hyphal elements were observed on the Gram stain of the submitted tracheal aspirate (Image 2A). Robust fungal growth was noted within 48 hours on Brain Heart Infusion and Inhibitor Mold agars from both the tracheal aspirate and bronchial wash specimens (Image 2B). A lactophenol cotton blue prep revealed broad hyphae with few septations, consistent with a member of the Mucorales (Image 3). Sporangiophores were noted to be long, dark and branched with round sporangia. Few rhizoids were observed and located between the sporangiophores. A definitive identification of Rhizomucor sp. was obtained through the use of matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS).

Image 2. A: Gram stain (400X) of direct sample from tracheal aspirate with broad, irregular, long, pauciseptate hyphae consistent with infection from a fungal species belonging to the order Mucorales. B: Fluffy “cotton candy” appearing light brown-grey colonies grew rapidly (<4 days) on brain heart infusion agar from both tracheal aspirate (pictured) and bronchial washing specimens.
Image 3. Lactophenol cotton blue stain obtained by tape preparation direct from fungal colony (A; 200X, B-D; 400X). Numerous branched dark brown non-apophysate sporangiophores with spherical columella were seen along with numerous broad, irregular pauciseptate hyphae with right angle branching (A, B). Rudimentary rhizoids developing off stolons (C, D) were rarely identified.

Discussion

Members of the order Mucorales can be identified in the laboratory by their rapid, robust growth and cotton candy-like appearance on conventional fungal media excluding those containing cycloheximide.2 Microscopically, these molds exhibit pauciseptate, broad (9-15 μm wide) hyphae with sporangia. Some species elaborate root-like structures called rhizoids (e.g Rhizopus, Rhizomucor) while others lack them (e.g. Mucor).2 Rhizomucor sp. can be differentiated microscopically from other related members of the Mucorales by its branched, dark brown sporangiophores with absent apophysis, round columella and the presence of few, short, rudimentary rhizoids (Image 3A-D). In practice, the rhizoids can be challenging to identify microscopically.2 Some species can also grow at elevated temperatures (~38-58°C) which can be utilized as a tool for use in identification.2 Newer methods including the use of MALDI-TOF MS and DNA probes allow for rapid, accurate identification of these fungi, but are not routinely available in all laboratories.

Infections caused by Mucorales (Mucormycosis) usually involve immunocompromised patients with defects in cell-mediated immunity. Rapid and often fatal, these infections can prove extraordinarily difficult to manage. They are known to be angioinvasive and can widely disseminate. Debridement of involved tissues and higher-level antifungal agents (e.g. posaconazole, amphotericin B) are mainstays of therapy. The most recognized group of patients where these infections are identified are uncontrolled diabetics, frequently in diabetic ketoacidosis, often with nasal/orbital sinus involvement. Patients with leukemia/lymphoma who are undergoing stem cell transplantation are another group often affected. In addition to the nasal/orbital sinuses, the gastrointestinal tract, skin and lungs also serve as important sites for these infections, especially if mucositis occurs.1

Rhizomucor sp. are an occasional cause of mucormycosis, but have a predilection for leukemic patients such as in this case.2 Given the significant bronchoscopy findings and the intraoperative presence of necrotic brain tissue, there was substantial clinical concern for invasive pulmonary mucormycosis with possible central nervous system involvement. Isavuconazole was discontinued, and posaconazole and micafungin were added to her antifungal therapy (L-AMB). Granulocyte infusions were used in an attempt to increase her cell-mediated immune response to the mold. Cardiothoracic surgery evaluated the patient but the lesion was deemed unresectable. Due to the presence of epistaxis, the otolaryngology service evaluated the patient for invasive fungal sinusitis; however, nasal endoscopy did not reveal any nasal/sinus involvement. The patient never regained significant neurological function and continued to medically decline during the hospitalization. She was placed on comfort care where she died shortly afterwards.

References

  1. Love GL, Ribes JA. 2018. Color Atlas of Mycology, An Illustrated Field Guide Based on Proficiency Testing. College of American Pathologists (CAP), p. 244-274
  2. Walsh TJ, Hayden RT, Larone DH. 2018. Larone’s Medically Important Fungi, A Guide to Identification. ASM Press, p. 185-190

-John Markantonis, DO is the current Medical Microbiology fellow at UT Southwestern and will be completing his Clinical Pathology residency in 2022. He is also interested in Transfusion Medicine and parasitic diseases.

Kim Stewart BS, MT(ASCP)SM holds a bachelor’s degree from Texas Tech University and is medical technologist in the microbiology section at UT Southwestern Medical Center with 35 years’ experience.

-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.

Microbiology Case Study: 68 Year Old Female with Streptococcus anginosus in a Tracheostomy Wound

Case History

A 68 year old female was admitted to our hospital for emergent tracheostomy due to airway obstruction. Her symptoms began months ago with cough, dysphagia, and hoarseness of voice, difficulty breathing, slowly growing neck mass, and weight loss. Surgical biopsy from the neck mass confirmed the presence of squamous cell carcinoma of the hypopharynx “stage IVB”.

Chest CT scan indicated the possibility of metastatic disease to the lungs. Recently, she experienced a slow onset of paraparesis and blurring of vision in her left eye, which raised a concern about the disease metastasized to the brain. Brain MRI was performed and came back negative for metastasis to the brain. Additionally, she is at risk of refeeding syndrome due to muscle wasting, cachexia, dysphagia, 25% body weight loss, and significant failure to thrive. Radiation oncologists recommended concurrent radiation with chemotherapy, with a prescribed 70 Gray of radiation dose.

Physical examinations showed bilateral coarse lung sounds. Prior to her presentation, she had been treated with docusate, hydrocodone-acetaminophen, morphine, ondansetron, and labetalol. Her past medical history was notable for essential hypertension and an extensive history of smoking 1 PPD for 45 years. She was also started on weekly carboplatin and paclitaxel for five weeks.

The surgical biopsy from the tracheostomy lesions was sent to the microbiology laboratory for bacterial culture. The Gram stain of the culture showed gram positive cocci in chains. After 48 hours of incubation, the cultures grew a pure culture of α-hemolytic colonies (Figure 1). The organism was identified as Streptococcus anginosus by MALDI-TOF mass spectrometry (VITEK MS).

Image 1. Alpha-hemolytic colonies of Streptococcus anginosus growing on Sheep Blood Agar (SBA) plate after 48 hours of incubation.

Discussion

Streptococcus anginosus group (SAG) “formerly named as streptococcus milleri” are members of the Viridians Streptococci group, which are known to cause endocarditis due to their ability to bind extracellular matrix proteins such as fibronectin, fibrinogen, and laminin, by facilitating bacterial adhesion to the heart valves.1

In general, SAG consists of three main strains: Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus. Streptococcus intermedius tends to be associated with central nervous system (CNS) infections, while S. anginosus is commonly found as commensals at the genitourinary and gastrointestinal systems.2 Since S. anginosus strains can be virulent due to its ability to survive in an acidic environment and cause systemic bacteremia, skin and soft tissue infections (SSTIs), osteomyelitis, and CNS infections, isolation of this organismfrom invasive sites should not be regarded as a contaminant. Besides, S. anginosus infection is occasionally associated with liver abscesses and colonic adenocarcinoma.3

Notably, the recovery of this organism from our patient’s tracheostomy biopsy wound indicates the likely association of S. anginosus and oral squamous cell carcinoma (OSCC). Sasaki M et al. demonstrated that the dental plaques in oral squamous cell carcinoma patients could act as a reservoir for SAG, which might cause significant DNA damage in oral mucosa, thus predisposing to accumulated mutations.4 Two other studies have also shown that SAG is recovered exclusively in oral squamous cell carcinoma patients compared to individuals without oropharyngeal cancer.5,6

SAG are usually susceptible to penicillin, ampicillin, or ceftriaxone, while sometimes they can be resistant to clindamycin.7 In our case, the patient received multiple doses of IV ampicillin-sulbactam and metronidazole in the emergency department. After S. anginosus had been identified from her tracheostomy wound, the patient was discharged on oral ampicillin-sulbactam, along with the carboplatin and paclitaxel treatment.

References

  1. Asam D, Spellerberg B. Molecular pathogenicity of Streptococcus anginosus. Mol Oral Microbiol. 2014;29(4):145-155. doi:10.1111/omi.12056
  2. Whiley RA, Beighton D, Winstanley TG, Fraser HY, Hardie JM. Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus (the Streptococcus milleri group): association with different body sites and clinical infections. J Clin Microbiol. 1992;30(1):243-244. doi:10.1128/JCM.30.1.243-244.1992.
  3. Masood U, Sharma A, Lowe D, Khan R, Manocha D. Colorectal Cancer Associated with Streptococcus anginosus Bacteremia and Liver Abscesses. Case Rep Gastroenterol. 2016;10(3):769-774. Published 2016 Dec 13. doi:10.1159/000452757
  4. Sasaki M, Yamaura C, Ohara-Nemoto Y, et al. Streptococcus anginosus infection in oral cancer and its infection route. Oral Dis. 2005;11(3):151-156. doi:10.1111/j.1601-0825.2005.01051.x
  5. Robayo DAG, Erira HAT, Jaimes FOG, Torres AM, Galindo AIC. Oropharyngeal Squamous Cell Carcinoma: Human Papilloma Virus Coinfection with Streptococcus anginosus. Braz Dent J. 2019;30(6):626-633. doi:10.1590/0103-6440201902805.
  6. Tateda M, Shiga K, Saijo S, Sone M, Hori T, Yokoyama J, Matsuura K, Takasaka T, Miyagi T. Streptococcus anginosus in head and neck squamous cell carcinoma: implication in carcinogenesis. Int J Mol Med. 2000 Dec;6(6):699-703. doi: 10.3892/ijmm.6.6.699. PMID: 11078831.
  7. Tracy M, Wanahita A, Shuhatovich Y, Goldsmith EA, Clarridge JE 3rd, Musher DM. Antibiotic susceptibilities of genetically characterized Streptococcus milleri group strains. Antimicrob Agents Chemother. 2001;45(5):1511-1514. doi:10.1128/AAC.45.5.1511-1514.2001.

-Ahmed Ismail Younes, MD., is a first year pathology (AP/CP) resident at the East Carolina University Brody School Medicine. He is interested in specializing in dermatopathology. He is also passionate about conducting translational research. In his spare time, Ahmed enjoys spending his time watching movies, baking homemade pizza, and playing soccer.

-Phyu M. Thwe, Ph.D., MLS (ASCP)CM is Technical Director/Technical Consultant at Vidant Medical Center Clinical Microbiology Laboratory. She completed a Clinical and Public Health Microbiology Fellowship through a CPEP-accredited program at the University of Texas Medical Branch (UTMB) in Galveston, Texas. She is interested in extrapulmonary tuberculosis pathophysiology and developing diagnostic algorithms.

The Rogue Anastomosis: Cytology Case Study

There are well over a hundred different cells types in the human body, and all those that have the ability to proliferate physiologically have the potential to succumb to uncontrolled cell division and thus generate a neoplasm. The tumors we most frequently encounter, like epithelial or hematologic disorders, are due to the higher proliferative rate of those cell types and the increased likelihood of an aberration or something going awry, i.e. mutations.2 Though we do come across many sarcomas and other mesenchymal tumors, this case study features a tumor derived from a cell type and structure that is rarely featured in cytology texts (an assumption, as I couldn’t find any information in the available prints within my department).

A 54 year old male patient presented to gastroenterology for severe cholecystitis. After undergoing a laparoscopic cholecystectomy, the pain subsided, but the patient was encouraged to follow up with a colonoscopy and upper endoscopy (EGD) due to his age. On the EGD, the gastroenterologist identified Barrett’s esophagus as well as a small, oval intramural lesion in the greater curvature of the gastric antrum, measuring 1.6 centimeters. The gastroenterologist was sharing the patient’s history with me when he described the endoscopic ultrasound findings of a well-defined hypoechoic and heterogenous lesion appearing to originate from the muscularis propria. His differential diagnoses based on imaging included a GIST, leiomyoma, or glomus tumor. The gastroenterologist did mention that the likelihood of this being a GIST or leiomyoma was very low. Despite lack of vascular structures visualized on Doppler imaging, the needle passes which I received to make air-dried Diff-Quik-stained smears were exceptionally bloody both grossly and microscopically.

I called my pathologist on cytology service for the day, informed him of the history presented to me by the gastroenterologist, and upon seeing the “rare atypical cells” through our telepathology platform, I hear him say, “a glomus tumor? Hmm, yes, it could be a glomus tumor.”
You know that feeling when you’ve asked someone to repeat themselves three times and you still didn’t comprehend them, so out of courtesy, you pretend to know what they said and express understanding? Yet internally you are confused beyond repair?

I repeated back to the gastroenterologist, “he said it could be a glomus tumor!”
Here comes the inner dialogue – what on earth is a glomus tumor? Why am I trying so hard to not look like an idiot right now? It’s okay to not know things, especially rare tumors that you’ve never come across. I have no idea what this tumor is, where it originates, what it consists of, this looks like a cohesive neuroendocrine tumor to me or even a basaloid squamous cell carcinoma. Obviously, it’s representative of the lesion, but I have no idea what a glomus (should I be pronouncing it with a long “o” or a short “o”?) is.

Images 1-2. Distal Gastric Antrum, Submucosa, FNA – DQ-stained smears.

After screening the Pap-stained smears and H&E Cell Block sections the next morning, the cells still had cuboidal cells with features similar to a neuroendocrine tumor to me. The best I could call it was a neoplasm with neuroendocrine features before leaving the case with the pathologist on service to order immunostains.

Images 3-5. Distal Gastric Antrum, Submucosa, FNA – 3-4: Pap-stained smears; 5: H&E cell block section

IHC returned that afternoon, showing the neoplastic cells to be positive for SMA, focal weakly positive for synaptophysin, and negative for PanCK, CD34, desmin, chromogranin, CD45, DOG-1, HMB-45, and S-100 protein. Between the immunostains and the morphology findings, the case was signed out as a glomus tumor. Additional immunostains were performed showing the tumor cells are positive for vimentin and have a Ki-67 proliferation index of only 1%. When the tumor was resected two months after the initial FNA, pathology reported the findings as a 1.0 centimeter glomus tumor that was completely excised.

Images 6-7. Distal Gastric Antrum, Submucosa, Resection – 6, H&E section 100X; 7, H&E section 400X.

This tumor arises from the glomus body, which is a normal arteriovenous shunt that aids in regulation of temperature and blood flow in the body. Surrounded by smooth muscle tissue, the glomus body contracts and relaxes, closing and opening the shunt between the efferent venules and the afferent arteriole to pull blood flow away from the periphery and back into the body’s core or to allow heat dissipation. Glomus tumors are most often found in the dermis of the fingertips and toes, especially under nail beds due to the pain and cold sensitivity from exposure to cold.1,5 Glomus bodies are also found in the stomach as a thermoregulator (think cold food/liquids entering the digestive system), and account for 1% of mesenchymal gastric tumors.4 Most glomus tumors are benign and rarely undergo malignant transformation, and complete excision of these tumors typically provides immediate relief with little to no chance of recurrence.1,3

References

  1. Fazwi, R., Chandran, P. A., & Ahmad, T. S. (2011). Glomus Tumour: A Retrospective Review of 15 Years Experience in A Single Institution. Malays Orthop J., 5(3), 8-12. doi:10.5704/MOJ.1111.007
  2. Holly, J. M. P., Zeng, L., & Perks, C. M. (2013). Epithelial cancers in the post-genomic era: should we reconsider our lifestyle? Cancer and Metastasis Reviews, 32(3–4), 673–705. https://doi.org/10.1007/s10555-013-9445-5
  3. Nascimento, E. F. R., Fonte, F. P., Mendonça, R. L., Nonose, R., de Souza, C. A. F., & Martinez, C. A. R. (2011). Glomus Tumor of the Stomach: A Rare Cause of Upper Gastrointestinal Bleeding. Case Reports in Surgery, 2011, 1–5. https://doi.org/10.1155/2011/371082
  4. Papadelis, A., Brooks, C. J., & Albaran, R. G. (2016). Gastric glomus tumor. Journal of Surgical Case Reports, 2016(11), rjw183. https://doi.org/10.1093/jscr/rjw183
  5. Uddin, M. M., Biswas, S. K., Rahman, M. H., Karmakar, N. C., Rahman, M. M., Alam, S. A. U., & Mondal, A. R. (2017). Sub-ungual Glomus Tumor: Study of 20 Cases. Faridpur Medical College Journal, 12(2), 64–67. https://doi.org/10.3329/fmcj.v12i2.34230

-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.

Transfusion Medicine Case Study: Fetal Maternal Hemorrhage in a Trauma Patient

A 31 year old woman, gravida 1 para 0, 35 weeks pregnant, arrived in the emergency room via ambulance following a fall down the stairs. The ER ordered a CBC, Type and Screen and a Kleihauer-Betke (KB) test and sent blood to the lab. The KB result was positive with 1.1 % fetal cells. Hypothetically, if this was an exam question, you might be asked, “How many doses of Rhogam should be administered?” But, before you grab your calculators, let’s explore that a bit.

Hemolytic Disease of the Fetus and Newborn (HDFN) has been described since the early 1600s, before blood groups were recognized. In the early 1900s, pioneers in blood banking, Landsteiner and Weiner, discovered the ABO and Rh blood groups, and, later, the Rh system became associated with HDFN. However, the antibody related etiology and pathogenesis of HDFN was not recognized until the late 1930s. Thus, the disease was written about in memoirs of midwives and physicians as early as 1609, but the mechanism involved was not described for another 300 years. The KB test was developed in 1957 by Enno Kleihauer and Klaus Betke to quantitate fetal maternal hemorrhage (FMH). The KB test allows physicians to diagnose and monitor and to initiate therapy to prevent the effects of HDFN. Finally, considered one of the most significant successes in medicine, prophylaxis for Rh HDFN, Rh immune globulin (RhIg), became available in 1968. The KB test is used to quantitate FMH in RhD negative mothers and the results can be used to calculate dosage for RhIg to prevent immunization. The KB test became one of the earliest examples of using a laboratory test to determine the appropriate dosage of a drug.1

KB testing has traditionally been used for RhD negative women to detect FMH and to determine the appropriate dose of RhIg to prevent immunization. In an RhD negative woman, we are concerned with immunization if the baby and mother are not antigenically similar. An RhD negative mother is given a prophylactic dose of RhIg at 28 weeks gestation. After delivery, when a newborn has a positive DAT and the fetal screen is positive, a quantitative test is needed to determine the appropriate dose of RhIg. In prenatal maternal trauma, there can also be a fetal bleed. Much as in childbirth, in a trauma, the baby’s blood can enter the mother’s circulation. This indicates placental hemorrhage and can be a prediction of preterm labor. In prenatal maternal trauma, the KB test has been used as aid in diagnosis and prognosis of HDFN, preterm labor and fetal demise. It can be used to determine if there has been a fetal bleed, and if so, to determine how much RhIg should be administered.

But, did you know that the KB test can also be used to determine FMH in RhD positive mothers? This is considered an alternative usage of the test. In the labs where I did KB tests, most fetal screens in Blood Bank were held until the following morning and performed on day shift. So, any KB tests on postpartum patients were also mostly done on day shift. I worked 2nd shift, and it was not uncommon to see KB tests ordered on RhD positive women. In fact, most of the KB tests ordered on 2nd and 3rd shift were from the ER and on RhD positive mothers. With RhD positive mothers, providers are not concerned with the mother producing anti-D, so RhIg is not a concern. Therefore, the answer to the hypothetical question posed above, is that this mother did not need any RhIg because, by checking the lab results it would be noted that this woman was Rh positive with a negative antibody screen.

A study performed in 2004 at the Shock Trauma Center, University of Maryland in Baltimore, reported that pregnant trauma patients with positive KB tests often had pre term contractions All patients in their study who experienced preterm contractions had positive KB tests. None of the patients with negative KB tests had uterine contractions. The conclusion was that “Kleihauer-Betke testing accurately predicts the risk of preterm labor after maternal trauma. Clinical assessment does not.” 2 They additionally concluded that, with a negative KB test, electronic fetal monitoring could safely be reduced. The major statement of the study, which has been incorporated into practice guidelines was that KB testing is important for all pregnant trauma patients, regardless of Rh status.2,3

In 2019 the College of American Pathologists Transfusion, Apheresis and Cellular Therapy Committee sent a survey with their proficiency testing program to determine how many participating laboratories perform KB tests on Rh positive pregnant females. 52% of the labs who responded noted that they performed quantitative fetal hemoglobin testing for RhD positive women, and about 39% reported performing more than 20 tests a year. The CAP group also reviewed literature detailing 16 observational studies and concluded that the literature supporting relying on the KB as a predictor of fetal distress was lacking evidence and nonconclusive. Despite the fact that doctors are ordering these and many laboratories are still performing this test STAT on RhD positive mothers, different guidelines for practice are mixed regarding if and how the KB should be used in these RhD positive trauma patients. Furthermore, many labs responded on the survey that doctors considered these results very important but that the labs were not sure how the results helped guide management of the mother or fetus.4

One of the problems some of these guidelines cite is that the KB test may not be rapid enough to use in trauma situations. Now, I have to start by saying that KB tests are probably no tech’s favorite test. The last hospital I worked at did KB tests in Hematology. Before that I worked at a hospital where we did KB tests in Blood Bank. There seems to be no way to avoid them! I would have to agree that a KB is not at all rapid. The test is both time sensitive, always ordered STAT, and very time consuming. Hands on time is considerable. I’ve gotten 2 in one night, on 2nd shift with only 4 or 5 techs manning the whole lab, and that makes for a busy night! Add a trauma or 2 to the mix, or a few units to wash for the NICU and you know why “Kleihaur-Betke” are not our favorite words.

Another concern is that the KB test is marketed as a quantitative test. The problem with this is that it is not very precise due to technical difficulty. In the KB acid elution test the mother’s blood is treated with acid and then stained and counterstained. Fetal cells contain HbF which is resistant to acid and these cells will remain bright pink. The mother’s cells, which are primarily HbA, will appear as faint ‘ghost’ cells. 2000 cells are counted and the percent of fetal cells is determined. The test is complicated and needs precision in staining, counting and calculations. A slide that’s too thick, poor timing of steps, slides that are not adequately dried, or fetal cells that fail to stain can all affect results and cause false negative results. In pregnant women HbF may be increased, and in women with hemoglobinopathies such as sickle cell anemia and thalassemia Hb-F can be increased, leading to false positive results. As well, late in pregnancy it would be considered normal to have some fetal cells in the mother’s circulation. Thus, both false negative and false positive KB results are not uncommon, and a positive report on a KB test may not accurately predict fetal distress.

Image 1. Kleihauer-Betke stain showing dark ink fetal cells and ‘ghost’ like pale maternal cells

In the CAP survey article, it was noted that, of participating labs, about 96% did KB tests and 4% use flow cytometry.4 Flow cytometry is accurate, sensitive and reliable for HbF determination. Flow cytometry uses antibodies directed against fetal hemoglobin and antibodies directed against adult RBCs. A clear separation of populations can be identified and quantitated. Despite the fact that it is well known that flow cytometry is a much more precise test for FMH, many laboratories continue to do KB testing. This is likely due to the fact that only a small percentage of labs have flow cytometers. If, in trauma situations, physicians want HbF determination with a “fast” turnaround time, KB testing can be done in house with no equipment necessary. This is not fast, but would, in most circumstances, be faster than sending a test to a reference lab.

The KB test has historically been validated and used to estimate the total amount of FMH, and the results used to calculate if additional doses of RhIg are indicated. The test has high specificity for HbF but can be subjective. Precision between techs and even with the same tech repeating the test can be relatively low. Because of this, the formula used to calculate RhIg dosage has a factor built in to make up for any imprecision. An alternate usage of the test, and the one used in this case example, is to predict outcomes and guide treatment in maternal trauma victims, regardless of Rh status.

While there is some controversy on using the KB test in these cases, it is none the less still recommended by many authors and included in medical guidelines.5 Providers are using the KB test more and more for assessing placental hemorrhage in cases of trauma and premature labor. Though immunophenotyping by flow cytometry has a greater accuracy, the KB test can give reliable results at a lower cost and with a faster turnaround time.

As always, this blog led me off on several tangents while writing. When I have an idea for a blog, I start with a case study or an interesting sample I have seen in the lab. The case study itself is the easy part, then I start researching and reading articles about the disorder, test or phenomenon that I am writing about. Often, when I read one article, I ask myself another question and say, “what if…?” and that leads to another article and another and another. Days later I can still find myself reading articles and chasing after more information. I love my job, I love being a Medical Laboratory Scientist and educator, and in true form of the curious MLS, I always want to investigate and never want to stop learning. Thus, this simple case about an alternative usage of Kleihauer-Betke (KB) test kept developing as I wrote. As a side note, it was interesting to see that the studies have had different conclusions and the guidelines for this use of the KB test have swayed over the years. It will be interesting to see what the future will bring. I have seen some articles about adding the HbF determination to hematology analyzers—wouldn’t that be nice!

References

  1. Reali G. Forty years of anti-D immunoprophylaxis. Blood Transfus. 2007;5(1):3-6. doi:10.2450/2007.0b18-06
  2. Muench MV, Baschat AA, Reddy UM, Mighty HE, Weiner CP, Scalea TM, et al. Kleinhauer-betke testing is important in all cases of maternal trauma. J Trauma 2004;57(5):1094-8.
  3. Michael V. Muench, Joseph C. Canterino, Trauma in Pregnancy, Obstetrics and Gynecology Clinics of North America, Volume 34, Issue 3, 2007, Pages 555-583.
  4. Matthew S. Karafin, Chad Glisch, et al, for the College of American Pathologists, Transfusion, Apheresis, and Cellular Therapy Committee; Use of Fetal Hemoglobin Quantitation for Rh-Positive Pregnant Females: A National Survey and Review of the Literature. Arch Pathol Lab Med 1 December 2019; 143 (12): 1539–1544.
  5. Krywko DM, Yarrarapu SNS, Shunkwiler SM. Kleihauer Betke Test. [Updated 2020 Sep 11]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan.

-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.

Microbiology Case Study: Interesting Case of a Cavitary Lung Mass

Case History

A 50 year old male with a significant past medical history of poorly controlled type 2 diabetes mellitus, hypertension, hyperlipidemia, smoking tobacco abuse and obstructive sleep apnea was referred to our institution’s pulmonology clinic for cavitary lung mass. The lung mass was incidentally discovered on chest x-ray and has been clinically stable on serial imaging for over two years; however, a previous extensive laboratory workup including computed tomography (CT) guided biopsy was unrevealing to its etiology. The patient was noted to be largely asymptomatic at his initial office visit; repeat diagnostic workup was ordered. CT chest imaging revealed a 2.8 x 1.9 x 2.0 cm cavitary lung mass in the posterior left lower lobe that was unchanged compared to outside CT imaging from approximately 4 months prior.

Image 1. Cross section (left) and Sagittal (right) views from CT chest without contrast revealed a 2.8 cm transverse by 1.9 cm anteroposterior by 2.0 cm craniocaudal stable mass-like opacity in the left lower lobe superior segment broadly abutting the posterior pleura with a tiny internal focus of cavitation.

Given the chronicity of the lung mass, atypical infection (Nocardia, endemic fungi, mycobacterium) and primary pulmonary cancer were highest on the differential diagnosis. Blood tests including endemic fungal serologies, QuantiFERON-TB Gold, cryptococcal antigen, galactomannan and Fungitell (1-3)-B-d glucan assay were negative. Given the unrevealing non-invasive workup, a repeat CT guided biopsy was performed and core biopsy samples were sent for AFB, fungal and Nocardia cultures as well as for histopathological examination.

Histopathology revealed necrotizing granulomatous inflammation with empty spherules of Coccidioides suggestive of a remote infection of long duration (Images 2, 3). Additionally, no microorganisms were isolated from cultures. Based on these findings, an infectious disease (ID) consult was placed. The patient remained asymptomatic and revealed a long history of residing within areas of the Southwestern United States endemic to Coccidioides species (sp.) during his ID office visit. Repeat Coccidioides complement fixation was positive for IgG (Titer: 1:4) with negative IgM by immunodiffusion testing. Urine Coccidioides antigen tested by quantitative sandwich enzyme immunoassay was negative. These findings likely represent past history of coccidiomycosis and not active infection. Antifungal therapy was deferred due to the patient’s asymptomatic status. The patient was monitored with close clinical follow up and continued serial imaging.

Histopathology Images

Image 2. Hematoxylin and eosin stained sections of formalin fixed paraffin embedded (FFPE) tissue from core biopsy of cavitary lung mass. Necrotizing granulomatous inflammation at 40X (A) and 100X (B) with empty spherules of Coccidioides (C, D) at 600X.
Image 3. Special stains performed of formalin fixed paraffin embedded (FFPE) tissue from core biopsy of cavitary lung mass highlighting empty spherules. Grocott’s methenamine silver stain at 100X (A) and 400X (B). Periodic Acid Schiff for Fungus stain at 600X (C).

Discussion

Coccidioides sp. are dimorphic fungi with a mycelial (saprophytic) phase in the environment and a spherule (parasitic) phase in its host.1 It is the cause of coccidiomycosis, also known as valley fever, desert fever or San Joaquin fever, which has a wide range of clinical presentations from subclinical manifestations (~60%) to an influenza-like illness followed by skin lesions to the most pathological form, disseminated disease.1 It can also cause the development of cavitary lung masses, as described in this case.1 It is endemic to the Southwestern region of the United States where it prefers dry, arid conditions.2 Infections normally occur by inhalation of infective arthroconidia, which have matured from mycelium, following disruption of soil.1 Once inside the host, lungs spherules containing endospores develop (Image 4).1 These spherules rupture releasing the endospores which can continue to develop into spherules to maintain a continuous parasitic cycle or can be exhaled into the environment to continue its saprophytic phase.1

Image 4. High magnification images of hematoxylin and eosin stained sections of formalin fixed paraffin embedded (FFPE) lung tissue revealing multiple spherules containing endospores (left) consistent with active Coccidioides infection and a giant ruptured spherule releasing endospores (right) that will continue to propagate Coccidioides infection.

Two morphologically indistinct species exist (C. immitis and C. posadasii) that can only be definitively identified by molecular methods.3 C. immitis is predominantly found in central and southern California while C. posadasii can be found in other non-Californian southwestern US states and extending into western Texas and down throughout Mexico and South America.3 When cultured, it grows rapidly at both 25°C and 37°C into woolly white colonies that develop alternating barrel-shaped arthroconidia that can be seen on tape prep with lactophenol blue.4

References

  1. Donovan FM, Shubitz L, Powell D, Orbach M, Frelinger J, Galgiani JM. 2019. Early Events in Coccidiomycosis. Clinical Microbiology Reviews, 33, e00112-19, DOI: 10.1128/CMR.00112-19
  2. Hernandez H, Erives VH, Martinez LR. 2019. Coccidioidomycosis: Epidemiology, Fungal Pathogenesis and Therapeutic Development. Current Tropical Medicine Reports, 6, 132-144,  DOI: 10.1007/s40475-019-00184-z
  3. Kirkland TN, Fierer J. 2018. Coccidioides immitis and posadasii; a review of their biology, genomics, pathogenesis, and host immunity, Virulence, 9:1, 1426-1435, DOI: 10.1080/21505594.2018.1509667
  4. Love GL, Ribes JA. 2018. Color Atlas of Mycology, An Illustrated Field Guide Based on Proficiency Testing. College of American Pathologists (CAP), p. 234-235

-John Markantonis is the current Medical Microbiology fellow at UT Southwestern and will be completing his Clinical Pathology residency in 2022. He is also interested in Transfusion Medicine and parasitic diseases.

-Dominick Cavuoti is a Professor at UT Southwestern and specializes in Infectious Diseases Pathology, Medical Microbiology and Cytology.

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

Hematology Case Study: A 20 Year Old with Anemia

Case History

A 20 year old Black male with a known history of HbS trait went to the primary care office for a pre-surgical evaluation for elective laparoscopic cholecystectomy for symptomatic cholelithiasis. All physical exam findings were negative. The patient had blood work completed and was found to have mild anemia with microcytosis. On previous imaging, the spleen was noted to be slightly enlarged. Further workup included a peripheral blood smear, finding target cells, microspherocytes, folded cells, and rod-shaped Hb C crystals (see image below). No sickled RBCs were noted.

Image 1. Peripheral blood smear with anemia, increased polychromatphilic RBCs, numerous target cells and rare HbC crystals

Discussion

Hemoglobin C disease is an intrinsic red cell disorder caused by Hemoglobin C (Hb C). Hb C is a variant of normal Hemoglobin A (Hb A) that results from a missense mutation in the β-globin protein, replacing the glutamic acid at position 6 with a lysine molecule. The disease can be either in the homozygous state (Hb CC) or in the heterozygous states (Hb AC or Hb SC). The origin of this mutation was traced back to West Africa and is found to confer protection against severe manifestations of malaria. In the United States, the Hb C allele is prevalent in about 1-2% of the African American population. There is an equal incidence between gender, and the incidence of the homozygous disease (i.e., Hb CC) is only 0.02%. Nevertheless, these statistics may be under-representative, since the disease is generally asymptomatic.

Heterozygous individuals with Hb AC usually show no symptoms, while homozygous individuals with Hb CC can have mild hemolytic anemia, jaundice, and splenomegaly. When Hb C is combined with other hemoglobinopathies, such as Hemoglobin S (Hb S), more serious complications can result. Hb S is similar to HbC in that it arises from a missense mutation; ie, a valine is substituted for the glutamic acid at the 6th position on the β-globin protein. As a result of this mutation, HbS abnormally polymerizes when in the presence of low oxygen tension, leaving the red blood cells (RBCs) rigid and irregularly shaped. Sickle cell disease (SCD) typically is a result of homozygous Hb S mutations (i.e., Hb SS), but the disease can also come from Hb SC.

All clinical features of Hb SS can be seen in Hb SC, including painful vaso-occlusive crises, chronic hemolytic anemia, stroke, acute chest syndrome, etc. Nevertheless, Hb SC is generally a milder disease. The complications from HbSC disease are less severe and less frequent when compared to Hb SS. Fortunately, unlike those with Hb SS disease, patients with Hb SC disease do not experience autosplenectomy, but they can develop splenomegaly. There are two complications that occur in HbSC disease occur more frequently than in HbSS disease, and they include proliferative sickle cell retinopathy and avascular necrosis of the femoral head (the latter case presents especially in peripartum women). Therefore, patients with HbSC disease should follow up with ophthalmology and obstetrics to monitor these complications. Furthermore, patients with Hb SC disease can vary in the severity of symptoms and the resulting complications. For example, some patients may develop a severe anemia and require blood transfusions; whereas, other patients are minimally affected by the disease. Overall, patients with Hb SC disease tend to have a better life expectancy compared to those with Hb SS disease. Patients with Hb SS disease have an average life expectancy of 40 years, while those with Hb SC disease are expected to live into their 60s and 70s. In contrast to Hb SS and Hb SC disease, Hb CC disease does not have an increase in mortality. As mentioned earlier, Hb CC disease results only in mild anemia, asymptomatic splenomegaly, and largely absent clinical symptoms.

Pathologic features of Hb SC and Hb CC diseases can be seen on a peripheral blood smear (PBS). Hb CC disease does not show sickled RBCs, while Hb SC can show sickled RBCs though very rarely. More importantly, Hb C is prone to polymerize into characteristic crystals. Depending on the zygosity of the individual, the crystals take on a defining shape. In heterozygous individuals (Hb SC), the crystals are found as irregular, amorphous, or bent appearing, and the RBCs can take on a “spiked and hooked” appearance. In homozygous individuals (Hb CC), the crystals are elongate, straight, and uniformly dense (as seen in the case above). In addition to crystals, the PBS shows numerous target cells, scattered folded cells, and microspherocytes.

Ancillary studies for diagnosis of these diseases include Hb variant analysis, such as electrophoresis and high-pressure liquid chromatography. Cellulose acetate (alkaline) electrophoresis is a standard method used to separate Hb A, Hb A2, Hb F, Hb C, Hb S, and other variants according to charge. Some hemoglobin variants comigrate using this described method, so citrate agar (acid) electrophoresis can be used additionally to distinguish between these variants. In Hb CC disease, analysis shows nearly all Hb C with small amounts of Hb F (i.e., fetal hemoglobin) and HbA2 (i.e., a normal variant of Hb A, in which the hemoglobin molecule is made up of 2 α chains and 2 δ chains). In Hb SC disease, analysis demonstrates almost equal amounts of Hb S and Hb C.

References

  1. Aster JC, Pozdnyakova O, Kutok JL. Hematopathology: A Volume in the High Yield Pathology Series. Philadelphia, PA: Saunders, an imprint of Elsevier Inc.; 2013.
  2. Gao J, Monaghan SA. Hematopathology. Chapter 1: Red Blood Cell/Hemoglobin Disorders. 3rd edition. Philadelphia, PA: Elsevier; 2018.
  3. Karna B, Jha SK, Al Zaabi E. Hemoglobin C Disease. 2020 Jun 9. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan–. PMID: 32644469.
  4. Mitton BA. Hemoglobin C Disease. Medscape, 9 Nov. 2019, emedicine.medscape.com/article/200853-overview.
  5. Saunthararajah Y, Vichinsky EP. Hematology: Basic Principles and Practice. Chapter 42: Sickle Cell Disease: Clinical Features and Management. Philadelphia, PA: Elsevier; 2018.

-Amy Brady is a 4th-year medical student at the Philadelphia College of Osteopathic Medicine. She is currently applying to AP/CP pathology residency programs. Follow her on Twitter @amybrady517.

-Kamran Mirza, MD PhD is an Associate Professor of Pathology and Laboratory Medicine and Medical Education, and the Vice-Chair of Education in the Department of Pathology at Loyola University Chicago Stritch School of Medicine. Follow him on Twitter @KMirza.

Hematology Case Study: Is it Pelger-Huët anomaly or Pseudo Pelger-Huët?

A 73 year old African American female had a CBC ordered as part of routine pre-op testing before knee surgery. The order for a CBC/auto differential and was run on our Sysmex XN-3000. CBC results were unremarkable, with the exception of a decreased platelet count. However, the instrument flagged “Suspect, Left shift?” and a slide was made for review. The CBC results are shown in Table 1 below.

Table 1. CBC results on 73 year old female.

Pelger-Huët anomaly (PHA), is a term familiar to medical laboratory professionals, but mostly from textbook images. PHA is considered to be rare, affecting about 1 in 6000 people. PHA has been found in persons of all ethnic groups and equally in men and women. The characteristic, morphologically abnormal neutrophils were first described by Dutch hematologist Pelger in 1928. He described neutrophils with dumbbell shaped, bi-lobed nuclei. The term ‘pince-nez’ has also been used to describe this spectacle shaped appearance. Pelger also noted that, in addition to hyposegmentation, there is an overly coarse clumping of nuclear chromatin. In 1931, Huët, a Dutch pediatrician, identified this anomaly as an inherited condition.

Pelger-Huët anomaly is an autosomal dominant disorder caused by a mutation in the lamina B receptor (LBR) gene on band 1q42. This defect is responsible for the abnormal routing of the heterochromatin and nuclear lamins, proteins that control the shape of the nuclear membrane.2 Because of this mutation, nuclear differentiation is impaired, resulting in white blood cells with fewer lobes or segments. In classic inherited PHA, cells are the size of mature neutrophils and have very clumped nuclear chromatin. About 60-90% of these neutrophils are bi-lobed either with a thin filament between the lobes, or without the filament. About 10-40% of total neutrophils in PHA have a single, non-lobulated nucleus. Occasional normal neutrophils with three-lobed nuclei may be seen.1 Despite their appearance, Pelger-Huët cells are considered mature cells, function normally and therefore can fight infection. It is considered a benign condition; affected individuals are healthy and no treatment is necessary for PHA.

Automated instruments may flag a left shift when they detect these Pelger-Huët cells. In this patient, the analyzer flagged a left shift and a slide was made and sent to CellaVision. The CellaVision pre-classified the Pelger-Huët cells as neutrophils, bands, and myelocytes. All of the neutrophil images were either bi-lobed or non-lobed forms. None of the neutrophils had more than 2 lobes. Eosinophils also had poorly differentiated nuclei. Cell images from this patient can be seen in Images 1-4.

Image 1. Images from CellaVision of bi-lobed “pince-nez” neutrophils with thin filament
Image 2. Non-terminally differentiated neutrophils pre-classified as bands on CellaVision. Bilobed variant without the thin filament.
Image 3. Non-lobed neutrophils with extremely coarse clumping of nuclear chromatin.
Image 4. Eosinophils in Pelger-Huët Anomaly.

If PHA is considered benign, with no clinical implications, why is it important to note these cells on a differential report? This slide was referred to our pathologist for a review. The patient had several previous CBC orders, but no differentials in our LIS. The pathologist reviewed the slide and, based on 100% of these neutrophils being affected, he reported “Pelger-Huët cells present. The presence of non-familial Pelger-Huët anomaly has been associated with medication effect, chronic infections and clonal myeloid neoplasms.” Thus, the importance of reporting this anomaly if seen on a slide. If the instrument flags a left shift, this is typically associated with infection. If these cells are misclassified as bands and immature granulocytes, with no mention of the morphology, there would be a false increase in bands reported and the patient may be unnecessarily worked up for sepsis.

An additional reason for reporting the presence of Pelger-Huët cells is that pelgeroid cells are also seen in a separate anomaly, called acquired or pseudo-Pelger-Huët anomaly (PPHA). PPHA is not inherited and can develop with acute or chronic myelogenous leukemia and in myelodysplastic syndrome. A type of PPHA may also be associated with infections or medications. Certain chemotherapy drugs, immunosuppressive drugs used after organ transplants, and even ibuprofen have been recognized as triggers for PPHA. PPHA caused by medications is typically transient and resolves after discontinuation of the drug. To add to causes, most recently, there have been studies published that report PPHA in COVID-19 patients.3

With several different causes of PHA/PPHA, a differential diagnosis is important. Is this a benign inherited condition, a drug reaction that will self-resolve after therapy is stopped, or something more serious? If Pelger-Huët cell are reported, it is important for the provider to correlate this finding with patient symptoms, treatments and history. There was no medication history and little other medical history in our case patient’s chart, and no mention of inherited PHA. The patient had also been tested for COVID-19 with her pre-op testing and was COVID negative. On initial identification of Pelger-Huët, a benign diagnosis that needs no treatment or work up would be the best outcome, so an attempt could be made to determine if the patient has inherited PHA. If other family members are known to have this anomaly, this would be the likely diagnosis as PHA is autosomal dominant. Family members can also easily be screened with CBC and manual differential. Molecular techniques are available to confirm PHA but are not routinely used. In the absence of this anomaly in other family members, it would need to be determined if the patient was on any medications that can cause pelgeroid cells. Inherited PHA and drug induced PPHA should be ruled out first because PPHA can also be predicative of possible development of CML or MDS. Considering this cause first could lead to unnecessary testing that might include a bone marrow aspirate and biopsy. Additionally, the entire clinical picture should be reviewed because in PPHA associated with myeloproliferative disorders there is usually accompanying anemia and thrombocytopenia and the % of pelgeroid cells tends to be lower.

Today most clinical laboratories have instruments that do automated differentials, and we encourage physicians to order these because they are very accurate and count thousands of cells compared to the 100 cells counted by a tech on a manual differential. Automated differentials are desirable for consistency and to improve turnaround times. Yet, it is important to know when a slide needs to be reviewed under the scope or with CellaVision. If a patient presents with a normal WBC and a left shift on the auto diff with no apparent reason, pictures can reveal important clinical information. Awareness of different causes of PHA/PPHA can relieve anxiety in patients and prevent extensive, unnecessary testing and invasive procedures.

References

  1. https://emedicine.medscape.com/article/957277-followup updated 8/4/2020
  2. Ayan MS, Abdelrahman AA, Khanal N, Elsallabi OS, Birch NC. Case of acquired or pseudo-Pelger-Huët anomaly. Oxf Med Case Reports. 2015;2015(4):248-250. Published 2015 Apr 1. doi:10.1093/omcr/omv025
  3. Alia Nazarullah, MD; Christine Liang, MD; Andrew Villarreal, MLS; Russell A. Higgins, MD; Daniel D. Mais, MD. Am J Clin Peripheral Blood Examination Findings in SARS-CoV-2 Infection . Pathol. 2020;154(3):319-329. 

-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.