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

Case History

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

Diagnosis

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

B1 FS: Frozen section remnants

B2-B6:     multiple representative sections from the cardia

B7-B10:   multiple representative sections from the body

B11-B12:   multiple representative sections from the pylorus

B13:     representative perpendicular section through the distal resection margin

B14:     seven putative lymph nodes

B15:     five putative lymph nodes

B16:     three putative lymph nodes

B17:     seven putative lymph nodes

B18:     six putative lymph nodes

B19:     three putative lymph nodes

B20:     six putative lymph nodes

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

Image 1.

Discussion

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

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

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

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

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

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

Image 2.

References

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

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

Microbiology Case Study: A 61 Year Old Male with Fevers and Weight Loss

Case History

The patient is a 61 year old male in good health until about 4 weeks prior to presentation when he sustained a tick bite on his left arm. He subsequently developed chills, fatigue, loss of appetite, and weight loss. Concerned that his symptoms were not improving, the patient presented to urgent care and a CBC was ordered. His CBC was remarkable for mild anemia (RBC count 3.96, HB 12.9) and thrombocytopenia (platelet count 78,000/cmm). Review of the peripheral blood smear revealed organisms present within his neutrophils. Given his history of a tick bite, Doxycycline was initiated for 14 days with immediate improvement of his symptoms, including a notable increase in appetite over the next few days.

Laboratory Identification

Image 1. Giemsa Stain showing a morulae within a neutrophil.

Within the neutrophils are purple organisms distinct from the nuclei identified as morulae. PCR testing for Anaplasma confirmed the result.

Discussion

 Anaplasmosis is a disease caused by the bacterium Anaplasma phagocytophilum, previously known as Ehrlichia phagocytophilum causing human granulocytic ehrlichiosis (HGE). A taxonomic change in 2001 identified that this organism belonged to the genus Anaplasma, and resulted in a change in the name of the disease to Anaplasmosis (1). These bacteria are obligate intracellular organisms in the Rickettsia family (1,2). Anaplasma cannot survive outside the cell and once it has been released, it rapidly induces uptake signals in other host cells (3). The number of Anaplasmosis cases reported to CDC has increased steadily since the disease became reportable, from 348 cases in 2000, to 5,762 in 2017(1).

Anaplasmosis is spread to people by tick bites primarily from the blacklegged tick (Ixodes scapularis) and the western black legged tick (Ixodes pacificus)(1,2). Anaplasmosis can be transmitted through blood transfusion and has been found in refrigerated blood more than a week after collection. Transfusion related infections have occurred from asymptomatic donors (1).

Signs and symptoms of Anaplasmosis typically begin within 1–2 weeks after the bite of an infected tick, which can be painless and often goes unnoticed. Early signs and symptoms (days 1-5) are usually mild or moderate and may include fever, chills, headache, muscle ache, nausea, vomiting, and lack of appetite (1,3). Rarely, if treatment is delayed or if there are other medical conditions present, Anaplasmosis can cause severe illness. Signs and symptoms of severe (late stage) illness can include respiratory failure, bleeding problems, organ failure, and death. Laboratory findings can include mild anemia, thrombocytopenia, leukopenia (characterized by relative and absolute lymphopenia and a left shift) and mild to moderate elevations in hepatic transaminases (1). Abnormal laboratory findings can appear in the first week of illness; however, normal laboratory findings do not rule out possible infection.

Co-infection with other tick borne illnesses such as Borrelia burgdorferi (Lyme disease), Babesia microti (Babesiosis), Ehrlichia muris eauclairensis (Erlichiosis), and Powassan virus can be seen so additional testing may be necessary in some patients. Methods for diagnosing Anaplasmosis include serology, molecular methods, and morphological identification. Though morphologic identification is extremely specific is lacks sensitivity making molecular methods such as PCR the diagnostic methods of choice (2). Treatment for most Rickettsial illnesses including Anaplasmosis are tetracyclines, especially doxycycline which is the drug of choice (1,3).

References

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

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

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

Microbiology Case Study: An 18 Year Old with Gastrointestinal Bleeding

An 18 year old female with no significant past medical history experienced multiple episodes of gastrointestinal bleeding over the course of a few weeks. The most recent bout included a bloody episode that filled the toilet, for which she provided a picture for the clinician. She denies any other associated symptoms including epigastric pain, nausea, vomiting, fever, or chills. Her travel history is unknown.

Review of her history reveals an unremarkable family and social history. She has never had an incident similar to this in the past and no other family members have ever complained of similar symptoms. Review of systems was unremarkable and within normal limits. Physical exam was unremarkable. A rectal exam was performed and was noted to have brown stool that was guaiac (occult blood) positive. Non bleeding internal hemorrhoids were noted. There were no external hemorrhoids present.

Labs drawn including CBC were within normal ranges with the exception of absolute eosinophils which were at the upper limit of normal range at 0.6 x 103/µL [normal range= 0.0 – 0.6 103/µL].

The patient had an esophagogastroduodenoscopy (EGD) to further investigate the gastrointestinal bleed. The exam was otherwise normal with exception of the ascending colon where they noted a worm on the surface of the mucosa (Image 1-2). The worm was collected and transported to microbiology for examination (Image 3-4).

Image 1. View of a worm seen on the mucosal surface of the ascending colon.
Image 2. Another view of a worm seen on the mucosal surface of the ascending colon.
Image 3. Adult worm viewed under the dissecting microscope.
Image 4. Eggs viewed under the dissecting microscope.

Discussion

Examination of the worm and eggs revealed morphology consistent with Trichuris trichiura, or whipworm.

T. trichiura is most prevalent in warm, moist regions. The worldwide prevalence of infection is estimated to be roughly 800 million, mostly among poorer populations. Infection from T. trichiura is spread via fecal-oral route and caused by ingesting embryonated eggs. This occurs when contaminated dirt is ingested or by consumption of vegetables or fruits that have not been carefully cooked, washed or peeled.

The male and female worms both have the long whip-like structures at the anterior end. T. trichiura worms are 30-50 mm in length and the average life span is 1 year but they can live up to 10 years. The females have a straight and thick head while the males have a curly ended head. The males are typically longer the females. The eggs classically have barreled shaped, brown eggs with thick shells that measure 50-55 µm long by 22-24 µm wide. At each pole is lucent mucoid plug. The can also vary in size as noted in Image 5.

The adult female T. trichiura produces 1,000-7,000 eggs per day. The life cycle begins as unembryonated eggs passed in feces into soil (Figure 1). It takes approximately 21 days in the soil for an unembryonated egg to go through the process of embryonation to become the infective form of the parasite. Once ingested, the embryonated eggs hatch in the human intestine.

Image 5. T. trichiura eggs (CDC DPDx website)
Figure 1. Lifecycle of T. trichiura (CDC, DPDx)

Clinically, symptoms vary depending on the worm biomass present with most infections being asymptomatic. Symptoms include cramping, weight loss, growth restriction in children, bloody stool, and anemia. It can also result in Trichuris dysentery syndrome, which is more common in children. Recurrent rectal prolapse has also been reported. Lab findings include peripheral eosinophilia. T. trichiura is treated with Albendazole for 5-7 days +/- Ivermectin. Our patient was then prescribed albendazole and is being followed in GI clinic.

References

  1. Centers for Disease Control and Prevention. “Laboratory Identification of Parasites of Public Health Concern: Trichuriasis”. https://www.cdc.gov/dpdx/trichuriasis/index.html
  2. Procop, G. W., Church, D. L., Hall, G. S., Janda, W. M., Koneman, E. W., Schreckenberger, P. C., & Woods, G. L. (2017). Koneman’s color atlas and textbook of diagnostic microbiology (Seventh edition.). Philadelphia: Wolters Kluwer Health.

-Sharif Nasr, MD, 4th year anatomic and clinical pathology resident at University of Chicago (NorthShore). Dr. Nasr has an interest in GI pathology.

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

Hematopathology Case Study: An 80 Year Old Man with Rapid Onset Cervical Adenopathy

Case History

An 80 year old man presented with rapid onset of cervical adenopathy over a period of few months. The largest lymph node measuring 6 cm was biopsied and sent for histopathological evaluation.

Biopsy Findings

Sections from the lymph node showed effacement of the lymph node architecture by a fairly monotonous population of medium to large sized lymphoid cells arranged in vague nodular pattern. Focally, a starry sky pattern was observed. The cells were 1.5-2 times the size of an RBC, with high N:C ratio, irregular angulated nuclei and small nucleoli. A high mitotic rate of 2-3 mitoses/hpf was seen.

Immunohistochemistry

Immunohistochemical stains showed that the lymphoma cells were positive for CD20, CD5, SOX-11, and negative for Cyclin D1, CD10, CD23, CD30, BCL-1, and BCL-6. Ki67 index was about 70%.

Diagnosis

A diagnosis of Mantle cell lymphoma, pleomorphic variant was made.

Discussion

Mantle cell lymphoma is a peripheral B cell lymphoma, occurring in middle aged or older adults, with a male: female ratio of 7:1. Although Cyclin D1 expression is considered a hallmark of mantle cell lymphoma, yet about 7% cases are known to be Cyclin D1 negative. In these cases, morphological features and SOX-11 positivity helps in establishing a definitive diagnosis.

Differential Diagnosis

In the assessment of morphological features of lymphoma, the cell size is an important starting point. In this case, the lymphoma cells ranged from medium to large sized. The following differential diagnoses were considered:

  • Burkitt lymphoma

This case showed a “starry sky” pattern focally. A medium sized population of cells, high mitotic rate and a high Ki67 index (70%) favoured a Burkitt lymphoma. However, although commonly seen in Burkitt lymphoma, a “starry sky” pattern is not specific for this type of lymphoma. Also, the lack of typical “squaring off” of nuclei, basophilic cytoplasmic rim were against the diagnosis of Burkitt lymphoma. The nuclei in this case showed 0-1 small nucleoli, unlike the typical basophilic 2-3 prominent nucleoli of Burkitt lymphoma. Moreover, Ki67 index, even though high was not enough for Burkitt lymphoma where it approaches 100%. The cells were negative for CD10 and Bcl-6, which are almost always found in a Burkitt lymphoma. Hence, a diagnosis of Burkitt lymphoma was ruled out.

  • Diffuse Large B cell Lymphoma

The presence of interspersed large cells with nucleoli, irregular nuclei, high mitotic rate, and a high Ki67 index with a history of very rapid enlargement of lymph node suggested a diagnosis of Diffuse Large B cell lymphoma. However, the scant cytoplasm, lack of bizarre cells, and absence of CD10, BCl-2, BCl-6 were against a diagnosis of DLBCL.

  • Lymphoblastic lymphoma

A diagnosis of lymphoblastic lymphoma was favoured by the irregularly angulated nuclei, and presence of nucleoli. However, the cells of lymphoblastic lymphoma have a more delicate nuclear chromatin, higher mitotic rate as against the relatively condensed chromatin and the low to high variable mitotic rate of Mantle cell lymphoma. Also, lymphoblastic lymphomas are more commonly of the T cell subtype and occur commonly in younger individuals. In this case, B cell markers were positive (CD 20), and the patient was 80 year old, disfavouring a lymphoblastic lymphoma. The blastoid variant of mantle cell lymphoma is practically indistinguishable from lymphoblastic lymphoma, except that it is Tdt negative.

Cyclin D1 negativity in Mantle cell lymphoma

In the cases of Cyclin D1 negative mantle cell lymphomas, morphology plays a critical role in coming to a diagnosis of mantle cell lymphomas. In this case, points that favoured the diagnosis of mantle cell lymphoma were clinical features such as older age (80 years), and male gender, and morphological features such as a vaguely nodular pattern of growth, irregular nuclei, and 0-1 small nucleoli. Due to the presence of variably sized cells with distinct nucleoli, a pleomorphic variant was considered. Even though Cyclin D1 was found to be negative, the cells were positive for SOX-11.

SOX-11 is a transcription factor that is not normally expressed in B cells, but is sensitive and fairly specific for mantle cell lymphomas. It is important to note that SOX-11 is also positive in 25% Burkitt lymphoma, 100% lymphoblastic lymphoma, and 66% T-prolymphocytic leukemia. Herein lies the importance of recognising morphological features, as all of these lymphomas that may express SOX-11 were ruled on the basis of morphology. A more specific antibody, MRQ-58 may be used for greater specificity. The presence of SOX-11 is considered a specific biomarker for Cyclin-D1 negative mantle cell lymphomas. In these cases, there is upregulation of Cyclin D2 or D3 that may substitute for Cyclin D1 upregulation. But, immunohistochemical detection of Cyclin D2 or D3 is not helpful for establishing a diagnosis, as other lymphomas are commonly positive for these markers. Hence, it is important to perform SOX-11 immunohistochemistry to diagnose the Cyclin D1 negative variant of mantle cell lymphoma.

SOX-11 can be used not just for the diagnosis, but also for determining prognosis of mantle cell lymphoma. Indolent MCL usually lack SOX-11 expression. The pattern of SOX-11 staining has also been used a marker of prognosis. Cytoplasmic expression of MCl, seen in only a few cases was associated with a shorter survival as compared to the more common nuclear staining of SOX-11.

Conclusion

In this age, lymphoma diagnosis relies heavily on the use of immunohistochemical markers. However, this case highlights the importance of morphological features in diagnosing lymphomas with unusual immunohistochemical marker profile. Although, this case was negative for Cyclin D1, considered a hallmark of Mantle cell lymphoma, yet, the combination of morphological features with SOX-11 staining helped in clinching the diagnosis. To avoid a misdiagnosis, it would be prudent to perform SOX-11 staining in all lymphoma cases morphologically resembling MCL, but lacking Cyclin-D1.

-Swati Bhardwaj, MD has a special interest in surgical pathology and hematopathology. Follow her on Twitter at @Bhardwaj_swat.

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

Microbiology Case Study: A 70 Year Old Male with Multiple Myeloma

Case History

The patient is a 70 year old male who was diagnosed with Kappa free light chain multiple myeloma. He was initially seen after he had a fall in the woods and underwent imaging which showed multiple lytic lesions and blood work showing monoclonal proteins and thrombocytopenia. He was found to have a lesion on his right scapula for which he received radiation. Bone marrow biopsy was performed which showed 60% plasma cells. To date he has completed radiation therapy, 5 cycles of chemotherapy, and is in the process of collecting stem cells for autologous stem cell transplant. Routine fungal culture of the stem cell collection grew a single tan white dry appearing colony on potato flake agar. A Gram stain of the organism revealed gram positive cocci mixed with filamentous structures.

Laboratory Identification

Image 1. Single tan white dry colony on potato flake agar.
Image 2. Modified acid fast stain (left) and Gram stain (right).
Image 3. Filamentous branching on Gram stain.

Based on the colony morphology and Gram stain results the organism was suspected to be in the Streptomyces genus. Identification with MALDI-TOF was attempted and did not yield a result as this bacteria is not in the data base.

Discussion

Streptomyces is a genus of gram positive aerobic saprophytic bacteria that grows in various environments, and has a filamentous form similar to fungi (1). The morphologic differentiation of Streptomyces involves identification of complex multicellular architecture with germinating spores that form hyphae, and multinuclear aerial mycelium, which forms septa at regular intervals, creating a chain of uninucleated spores (2,3). They are able to metabolize many different compounds including sugars, alcohols, amino acids, and aromatic compounds by producing extracellular hydrolytic enzymes (helping with degradation of organic matter). Their metabolic diversity is due to their extremely large genome which has hundreds of transcription factors that control gene expression, allowing them to respond to specific needs (3).

Streptomyces is also considered to be one of the most medically important bacteria because of its ability to produce bioactive secondary metabolites. These metabolites are used in the creation of antifungals, antivirals, antitumoral, anti-hypertensives, and many antibiotics and immunosuppressives. They are responsible for 2/3 of all the worlds naturally occurring antibiotics (1).

Streptomyces is usually considered a laboratory contaminant though they can cause infections in immunocompromised patients and are chiefly responsible for granulomatous lesions in skin also known as actinomycotic mycetomas (1,2). Invasive pulmonary disease has been seen in HIV patients, splenectomized patients with sarcoid, and rarely in immunocompetent hosts (1). More rare presentations include brain abscesses can be seen in patients with cerebral trauma, peritoneal infections have been shown to occur in patients undergoing multiple pericenteses, and bacteremia in patients with indwelling catheters (1). Infection with Streptomyces is not common so susceptibility data is limited. Available data shows that organisms were consistently susceptible to amikacin; frequently susceptible to imipenem, clarithromycin or erythromycin, minocycline, and trimethoprim-sulfamethoxazole; and infrequently susceptible to ciprofloxacin and ampicillin (4).

Our patient had not received the stem cell unit that this grew from, so another aliquot was requested. The second aliquot did not grow any organisms, so the Streptomyces was considered a contaminant.

References

  1. Procop, Gary W., et al. Konemans Color Atlas and Textbook of Diagnostic Microbiology. 7th ed., Wolters Kluwer Health, 2017.
  2. Tille, Patricia M. Bailey & Scotts Diagnostic Microbiology. 13th ed., Elsevier, 2014.
  3. Chater KF. Recent advances in understanding Streptomyces. F1000Res. 2016;5:2795. Published 2016 Nov 30. doi:10.12688/f1000research.9534.1
  4. Mona Kapadia, Kenneth V.I. Rolston, Xiang Y. Han, Invasive Streptomyces Infections: Six Cases and Literature Review, American Journal of Clinical Pathology, Volume 127, Issue 4, April 2007, Pages 619–624, https://doi.org/10.1309/QJEBXP0BCGR54L15

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

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

Microbiology Case Study: A 40 Year Old Woman with Fever, Chills, and Leg Pain

Clinical History

A 40 year old African American female with a history of sickle cell disease presented to an outpatient clinic with fever, chills, and leg and back pain consistent with a sickle cell crisis. Her past medical history was also significant for asthma and seizures. She rated her pain as 10 out of 10, her vitals showed a temperature of 101.0°F, and she was also tachycardic and hypotensive. Her white blood cell count was 23.0 TH/cm2, hemoglobin 8.4 g/dL, hematocrit 26.0%, and platelets 619,000 TH/cm2. In clinic, she received pain medications and a fluid bolus, two sets of blood cultures were collected, and she was transferred to the emergency department for further work up.

Laboratory Identification

Image 1. Gram stain from a positive blood culture bottle showing small, gram positive budding yeast (1000x oil immersion).
Image 2. A mucoid, salmon-colored yeast grew on Sabouraud dextrose and chocolate agars.

Blood culture bottles were positive after approximately two days on the automated instrument. The Gram stain showed small, gram positive budding yeast (Image 1). The BioFire FilmArray for blood culture identification was negative for Candida albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis. At this time, she was started on micafungin for antifungal therapy. A mucoid, salmon colored yeast grew on both Sabouraud dextrose and chocolate agars (Image 2) and was identified by Vitek 2 as Rhodotorula spp.

Discussion

Rhodotorula spp. are basidiomycetous yeasts that make up the normal microbiota on moist skin and can be found in bathtubs and on shower curtains. Rhodotorula spp. are usually considered contaminants, but can rarely cause fungemia in patients with central lines, endocarditis, peritonitis, and meningitis, especially in those that are immunocompromised. R. mucilaginosa, R. glutinis, and R. minuta are the species commonly associated with human disease. 

In the laboratory, Rhodotorula spp. grow as a mucoid, salmon colored yeast within 1-3 days of incubation. On Gram stain or lactophenol cotton blue prep, the yeast is small and round to oval with multilateral budding. Pseudohyphae are not usually present. Rhodotorula spp. produce urease and fail to ferment carbohydrates. R. mucilaginosa is negative for nitrate assimilation. Identification can also be confirmed by commercial kits, automated systems, and MALDI-TOF mass spectrometry. Rhodotorula spp. are intrinsically resistant to echinocandins and fluconazole.

In the case of our patient, she was switched to intravenous amphotericin B after the identification of Rhodotorula spp. was made. Reference laboratory testing identified the isolate as R. mucilaginosa with high minimum inhibitory concentrations (MIC) to fluconazole and echinocandins. Amphotericin had an MIC of 0.5 µg/ml. She successfully completed a 14 day course with close monitoring of creatinine, electrolytes, and platelet count. Repeat blood cultures were negative and no other focuses of infection were found on CT scans, transthoracic echocardiogram, and ophthalmology exam.

-Lisa Stempak, MD, is an Assistant Professor of Pathology at the University of Mississippi Medical Center in Jackson, MS. She is certified by the American Board of Pathology in Anatomic and Clinical Pathology as well as Medical Microbiology. She is the Director of Clinical Pathology as well as the Microbiology and Serology Laboratories. Her interests include infectious disease histology, process and quality improvement, and resident education.

Hematology Case Study: Thrombocytopenia in a 4 Year Old Child

A 4 year old child was brought to the pediatrician by her mother with a complaint of new onset of severe bruising on her legs. The mother could not recall any falls or bumps that would have caused the bruising. On exam, the physician also noted mucosal bleeding in the oral cavity. Questioning revealed that the patient had experienced flu like symptoms several weeks earlier. The physical exam was normal except for the bleeding. There was no family history of bleeding disorders. A CBC was ordered.

Reported CBC Results

WBC, RBC, Hgb, Hct, RBC indicies normal

Platelet count 26 x 103/μL

IPF 22% (reference range IPF% 1.0-7.0%) The physician evaluated the results, noting the normal CBC but decreased platelet count. The above results also show the immature platelet fraction (IPF), an additional Advanced Clinical Parameter reported from the Sysmex XN hematology analyzer. A low platelet count, as seen in this patient, will reflex a fluorescent platelet count (PLT-F). The impedance count (PLT-I) can be falsely increased if small RBCs or fragments are counted as platelets. On the other hand, in an optical platelet count, when measuring platelets by size (PLT-O), large platelets can be missed, giving a falsely low count. In this case there was a low platelet count and an instrument flag for an abnormal platelet scattergram. The PLT-F, on the other hand, uses a platelet specific dye which eliminates interference seen with other methods. The fluorescent dye labels the RNA, and forward scatter is used to determine size while side fluorescence is used to measure RNA content. With gating set based on cell volume and RNA content, the PLT-F can be measured. Therefore, the reflexed and more reliable PLT-F was the reported count.

Figure 1. PLT-F scattergram. The PLT-F channel measures forward scatter (FSC) on the Y axis and side fluorescence (SFL) on the X axis.1

Additionally, when there is an abnormal scattergram or a low platelet count, the IPF% and IPF# are also reported. The immature platelet fraction is a measure of the youngest platelets, or reticulated platelets. These are the first circulating platelets, right out of the bone marrow. An increased IPF indicates an increase in platelet production, yet this child’s platelet count was very low. This suggests that the thrombocytopenia may be due to excessive destruction of platelets; the bone marrow was actively making platelets, but they were being destroyed, causing the low platelet count.

Figure 2. Platelet scattergrams from a healthy individual with a normal IPF (a) and a patient with a high IPF (b). Mature platelets appear as blue dots, green dots represent the IPF with increased cell volume and higher fluorescence intensity compared to mature platelets.1

Diagnosis

Immune Thrombocytopenia- ITP.

Primary immune thrombocytopenia (ITP), formerly known as idiopathic thrombocytopenic purpura or immune thrombocytopenic purpura, is one of the most common bleeding disorders of children. In most cases, it presents with sudden onset of bruising and petechiae in an otherwise healthy child, with normal WBC and hemoglobin. ITP is an autoimmune bleeding disorder in which the immune system makes anti-platelet antibodies which bind to platelets and cause destruction. Even though the exact cause of ITP remains unknown, it is recognized that it can follow a viral infection or live vaccinations. While there are some similarities between pediatric ITP and ITP in adults, in children this tends to be an acute disease which is self-limiting and resolves itself in several weeks, with no treatment. However, in a small number of children, the disorder may progress to a chronic ITP. In contrast to ITP in children, a chronic form is more commonly seen in adults. It is usually a diagnosis of exclusion, does not follow a viral illness and requires treatment.

This patient recovered in a few weeks. One month after the initial episode, her PLT was 174 x 103/μL and her IPF% was 6.0%

Conclusion

An IPF reported with a CBC, in combination with a low platelet count, is fast, inexpensive, and can be extremely beneficial in aiding in a timely diagnosis. As the child’s platelet count recovered, the IPF% returned to normal range. ITP can therefore be monitored with a CBC. Thus, the IPF can be used not only to help diagnose but also as an indicator of remission.

References

  1. Sysmex America, 2019. www.sysmex.com/us. Used with permission
  2. Arshi Naz et al. Importance of Immature platelet Fraction as a predictor of immune thrombocytopenic purpura. Pak J Med Sci 2016 Vol 32 No 3:575-579
  3. Briggs,C. Assessment of an immature plateletfraction (IPF) in peripheral thrombocytopenia. Br J Haematol 2004Jul;126(1):93-9
  4. Sysmex White Paper. The role of the ImmaturePlatelet Fraction(IPF) in the differential diagnosis of thrombocytopenia. www.sysmex.com/us
  5. D-Orazio, JA, Neely, J, Farhoudi,N. ITP in children: pathophysiology and current treatment approaches.J Pediatr Hematol Oncol.2013 Jan;35(1): 1-13

-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.