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


  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.
  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.
  4. Papadelis, A., Brooks, C. J., & Albaran, R. G. (2016). Gastric glomus tumor. Journal of Surgical Case Reports, 2016(11), 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.

-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!


  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).


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


  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


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.


  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,
  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.


  1. 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.

Cytology Case Study: Strike a Chord

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

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

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

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

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

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

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

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

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


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

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

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

Case History

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

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

Laboratory Identification

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

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


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

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

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


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

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

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

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

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

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

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

Microbiology Case Study: 83 Year Old Female with a Perisplenic Abscess

Case History

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

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

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

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

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


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

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

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


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

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

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

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

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

Image 1. Lymphocytes with intracytoplasmic inclusions.

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

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

Image 2. Lymphocytes with crystalline-like inclusions.

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

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

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

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

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

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

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


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

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

Microbiology Case Study: 35 Year Old Male with Chest Pain

Case History

A 35 year old man presented to the Emergency Department (ED) with intermittent chest pain for 3-4 days, abdominal pain, fatigue, and lightheadedness over the same time period. Additionally, his family reported symptoms of progressive malaise for about a month, worse over the last week. In the ED, he was found to have ST elevations in the inferior leads of the electrocardiogram, which can be indicative of a heart attack. He was given 325 mg of aspirin and was emergently taken to the catheterization lab. He was found to have multiple complete occlusions in the distal left anterior descending artery (LAD), posterior descending artery (PDA), and posterior left ventricular artery (PLV). He underwent aspiration thrombectomy and the resulting clots were thought to be emboli; segments were sent to pathology for histopathologic evaluation and to microbiology for culture. There was no evidence of underlying plaque. He was admitted for management of ST-elevation myocardial infarction (STEMI). While in the ED, he was found to have white blood cell count of 23,000 and tachycardia to 110 beats per minute. A transthoracic echocardiogram demonstrated thickened aortic valve leaflets with evidence of leaflet destruction, severe aortic insufficiency, and right coronary cusp perforation which are consistent with endocarditis. Blood cultures were obtained and he was started on broad spectrum antibiotics (Vancomycin and Cefepime).

He has a past medical history significant for previous shoulder abscess with Methicillin-resistant Staphylococcus aureus (MRSA) and intravenous drug use (IVDU) (heroin, last use ~6 days prior to admission).

Computed tomography (CT) of his abdomen and pelvis revealed multiple renal infarctions and a splenic infarction (Image 1). In addition, the CT of the brain showed: “Multifocal scattered supratentorial and infratentorial subarachnoid hemorrhages and findings suggestive of evolving ischemic infarct involving the right inferior frontal gyrus, without evidence of hemorrhagic transformation currently. No midline shift or other complication identified.”

Image 1. Computed tomography of the abdomen demonstrating multiple renal infarctions (left, circled) and a splenic infarction (right, circled).

On hospital day 1 (HD1), both sets of initial blood cultures turned positive with gram positive cocci (GPC) in clusters and thrombectomy cultures were also growing GPC in clusters (Image 2). On HD2, the GPC in the thrombectomy culture was identified as Rothia mucilaginosa. GPC growing in the blood cultures were also Rothia mucilaginosa (Image 2). The patient was continued on Vancomycin. Repeat blood cultures were obtained after catheterization on HD0, and HD2, which were negative. On HD2, the pathology of the initial clots showed “fibrinopurulent debris and fibrin plaques with innumerable cocci in clusters” (Image 3).

Image 2. Microscopic and culture morphology of Rothia mucilaginosa. Left: Gram stain from a blood culture demonstrating groups of Gram-positive cocci in small clusters (1000x magfication, oil immersion). Right: Blood agar plate with mucoid light pink-gray colonies.
Image 3. Hematoxylin and eosin stained slide of formalin fixed paraffin embedded tissue of the thrombus removed during the initial emergent catheterization procedure. Sections demonstrate fibrinous material with entrapped white cells and innumerable cocci. Top: 100x magnification; Bottom: 400x magnification.

On HD3, the patient developed 10/10 chest pain with troponin elevation and T-wave inversion. He was taken back to the catheterization lab for another procedure where he was found to have recurrent, complete occlusion of the PDA with unsuccessful recanalization due to the dense thrombus. On HD6, he developed tamponade physiology due to a large pericardial effusion that was drained. Cultures of the pericardial fluid were negative. Given the recurrent embolization events, the patient was transferred to another hospital to undergo aortic valve replacement surgery and coronary artery bypass graft surgery. Cultures taken at the time of the valve replacement surgery were negative and the valve tissue was not sent for pathologic evaluation.  


We present an uncommon case of extensive Rothia mucilaginosa sepsis with septic emboli and endocarditis. Rothia mucilaginosa has experienced the scientific name-change game over the last several decades. It was first identified as Micrococcus mucilaginosus, then became Stomatococcus mucilaginosus, was also known as Staphylococcus salivarius before finally arriving to today’s name of Rothia mucilaginosa.1,2 R. mucilaginosa is a normal inhabitant of the oropharynx and is often associated with dental caries.3 R. mucilaginosa can cause invasive infections, typically in patients with compromised immune systems, disrupted mucosal barriers or injection drug use.4

R. mucilaginosa is a facultatively anaerobic, gram positive, non-fastidious coccus that is coagulase negative but with variable catalase positivity. Colony morphology is usually white to gray nonhemolytic colonies with a mucoid appearance. Although the variable catalase reaction may point toward a Streptococcus spp., the Gram stain morphology of clusters helps to rule it out. Although not all strains are mucoid, the classic colony morphology is wet and is due to polysaccharide capsule.

The organism is generally susceptible to antibiotics designed to target gram positive bacteria including, penicillin, ampicillin, cefotaxime, rifampin and vancomycin.4 It is important to note that R. mucilaginosa is not predictably susceptible to clindamycin, trimethoprim-sulfamethoxazole or ciprofloxacin.5 The patient presented in this case received intravenous vancomycin in part due to the extensive disease on presentation, but also because he was at risk for methicillin-resistant Staphylococcus aureus (MRSA) sepsis and had a previously documented abscess from MRSA.


  1. Bergan T, Kocur M. 1982. Stomatococcus mucilaginosus gen. nov., sp.nov., ep. Rev., a member of the family Micrococcaceae. Int. J. Syst. Bacteriol. 32:374-377
  2. Collins MD, Hutson RA, Baverud V, Falsen E. 2000. Characterization of a Rothia-like organism from a mouse: description of Rothia nasimurium sp.nov. and reclassification of Stomatococcus mucilaginosus as Rothia mucilaginosa comb.nov. Int. J. Syst. Evol. Microbiol. 3:1247-1251.
  3. Trivedi MN, Malhotra P. Rothia prosthetic knee joint infection. 2015. J. Microbiol. Immunol. Infect. 48(4):453-455.
  4. Bruminhent J, Tokarczyk MJ, Jungkind D, DeSimone JA. Rothia mucilaginosa Prosthetic Device Infections: A Case of Prosthetic Valve Endocarditis. J. Clin. Microbiol. 5;15:1629-1632.
  5. Kaasch AJ, Saxler G, Seifert H. 2011. Septic arthritis due to Rothia mucilaginosa. Infection. 39:81-82.

-Doreen Palsgrove, MD is a board certified Anatomic and Clinical Pathologist who joined the faculty at UT Southwestern as an Assistant Professor in 2019. She specializes in head and neck and genitourinary pathology. 

Dominick Cavuoti, DO is a professor of AP and CP at UT Southwestern, specializing in infectious disease pathology, cytology and medical microbiology.

-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.