“candida albicans” and “ovarian cancer” · candida albicans is an endogenous commensal...

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Cell Identification

VB

F-0

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Participants

Identification No. % Evaluation

Erythrocyte, mature 400 98.8 Educational

Macrophage containing abundant small lipid vacuoles/droplets (Lipophage)

3 0.7 Educational

Erythrocyte, nucleated 1 0.3 Educational

Starch granulate 1 0.3 Educational

The single view of the arrowed cell is a mature erythrocyte, as correctly identified by 98.8% of

participants. Because of the inconsistency of peritoneal fluid chemistry and the slide preparation

techniques, erythrocytes can have variable morphology in these specimens. Review of the entire slide

will reveal RBC fragments as well as more easily recognizable intact erythrocytes. Some erythrocytes

are present normally even in a specimen such as this peritoneal dialysate. Grossly bloody specimens

may indicate an intra-abdominal bleed and are cause for concern.

4

VB

F-0

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Participants


Yeast/fungi, extracellular 400 98.8 Educational


Bacteria - extracellular 1 0.3 Educational

Pneumocystis jirovecii 1 0.3 Educational

The two views with the arrowed organisms are yeast, specifically Candida albicans, as correctly

identified by 98.8% of participants. Candida albicans is an endogenous commensal organism that is part

of the normal flora of the human mucus membranes, gastrointestinal tract, respiratory tract, and vagina.

Candida albicans produces oval yeast forms, 5-7 µm in diameter, as well as elongated cells, referred to

as pseudohyphae, that can be up to 50 µm in length.

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VB

F-0

4

Participants


Neutrophil, segmented or band 396 97.8 Educational

Neutrophil/macrophage with phagocytized bacteria

3 0.7 Educational

Mesothelial cell 2 0.5 Educational

Eosinophil, any stage 1 0.3 Educational

Malignant cell (non-hematopoietic) 1 0.3 Educational

Monocyte/macrophage 1 0.3 Educational


The single view with the arrowed cell is a neutrophil, as correctly identified by 97.8% of participants. The

mature neutrophil shows the characteristic nuclear segmentation with thin condensed chromatin

filaments connecting the lobes. The cytoplasm contains fine pale pink granules filling most of the

cytoplasm, which is otherwise pale blue. Neutrophils are commonly identified in fluids from patients with

infections and inflammatory conditions.

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VB

F-0

5

Participants


Neutrophil/macrophage with

phagocytized fungi 395 97.5 Educational


3 0.7 Educational



Macrophage containing neutrophil(s) (Neutrophage)

1 0.3 Educational


The arrowed cells are neutrophils with phagocytized yeast as correctly identified by 97.5% of

participants. The intracellular yeast forms distort the cytoplasm and nuclear structures and are

morphologically similar to the free floating yeast forms surrounding these cells.

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VB

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Participants


Degenerating cell, NOS 365 90.1 Educational

Stain precipitate 29 7.2 Educational


Basophil, mast cell 2 0.5 Educational

Immature or abnormal call, would refer for identification

2 0.5 Educational


Eosinophil, any stage 1 0.3 Educational

Synoviocyte (synovial lining cell) 1 0.3 Educational

The arrowed cell is a degenerating cell as correctly identified by 90.1% of participants. The cell of

origin is unclear. Attempts to discern its lineage are rarely helpful in these cases and can often be

misleading. Degeneration is often seen in infection-related fluids due to the exuberant inflammatory

response and concomitant release of caustic enzymes.

Michael Crossey, MD, PhD

Hematology and Clinical Microscopy Resource Committee

8

Case Discussion for VBF-01 – VBF-06

This case illustrates Candida albicans peritonitis in a patient with renal failure who is on peritoneal dialysis.

Chronic renal failure patients maintained on dialysis, either hemodialysis or peritoneal dialysis, have a marked

increase in incidence of fungal infections, at over 9 times that of the general population. Most of these infections

are due to Candida species and the majority of these fungal infections are associated with peritoneal dialysis.

In kidney failure, or end-stage renal disease, the kidneys have lost the capacity to filter the blood and remove

waste and excess salt and water. Treatment options for these patients include hemodialysis, peritoneal dialysis,

and renal transplantation.

Hemodialysis requires the surgical creation of an arteriovenous fistula, an arteriovenous graft, or the insertion of a

central venous catheter to provide blood access for periodic dialysis, either at home, or at a hospital clinic. During

dialysis, blood is pumped from the body, filtered and returned to the body. When performed at a center, patients

usually go 3 times a week for 3 to 5 hours. Home dialysis is often done more frequently at 3 to 7 times a week for

3 to 4 hours or overnight.

Peritoneal dialysis requires the surgical insertion of a catheter in the abdominal cavity through which dialysis fluid

can be introduced and drained from the peritoneum. The dialysis fluid, or dialysate, is piped into the abdomen

where it stays for a period of time collecting waste and excess salt and water. It is then drained and clean

dialysate fluid is introduced. Patients usually perform 3 to 5 exchanges a day in this continuous ambulatory

peritoneal dialysis. Continuous cycling peritoneal dialysis uses a machine and is usually performed overnight.

The main complication of peritoneal dialysis is infection of the peritoneal space, or peritonitis. This is most

commonly caused by staphylococcal species. Fungal peritonitis occurs in 2 to 13 percent of cases and most of

these infections are due to Candida species, especially Candida albicans and Candida parapsilosis, but also

Candida krusei and Candida glabrata. Aspergillus, Fusarium, Rhodotorula, zygomycetes and dematiaceous

molds are sometimes the cause. A mixed bacterial and fungal infection can also occur; in these cases, response

to therapy may be poor.

Causes and risk factors for fungal peritonitis in patients on peritoneal dialysis include:

Breaks in sterile technique when connecting peritoneal catheters to dialysate bag

Skin infection of the catheter entry site

Intestinal perforation

Peritoneovaginal fistula

Migration of fungi across the bowel wall into the peritoneum

Recent use of antibiotics

Recent bacterial peritonitis

Emergency peritoneal dialysis

Human immunodeficiency virus (HIV) infection

Extraperitoneal fungal infection

Abdominal surgery

Contaminated water baths to warm dialysis fluid

Contact with pigeon guano

Contact with soil during gardening

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Patients with peritonitis, whether bacterial or fungal, present with abdominal pain, nausea, vomiting, diarrhea and

fever. The dialysate fluid will become cloudy, and, in infections due to molds, the catheter port may become

blocked.

In cases of suspected peritonitis, the peritoneal dialysate fluid should be examined in the laboratory. In addition,

peritoneal lavage may be used to diagnose acute peritonitis. This is performed by infusing 1.0 L of saline or

Ringer’s solution into the peritoneum and retrieving the fluid by gravity drainage. In peritoneal dialysis fluid, a

leukocyte count greater than 100 cells µL (0.1 x 109/L) with > 50 percent neutrophils is consistent with infection.

In lavage fluids, a WBC count of 200 cells/µL (0.2 x109/L) is associated with a 99% probability of peritonitis.

Fungal infections usually show a white blood cell count greater than 200 cells/µL (0.2 x109/L) with a

predominance of neutrophils. Peritoneal eosinophilia of greater than 10% is commonly associated with chronic

peritoneal dialysis in the absence of infection, but has been reported as a possible indicator of fungal peritonitis.

Fungal and yeast forms can be detected on routine Wright-Giemsa stained slides. In addition, gram stain of the

peritoneal fluid or dialysate fluid will demonstrate the yeast forms of Candida species. Some laboratories utilize

the fluorescent calcofluor white stain to demonstrate fungal organisms. To provide optimal sensitivity, at least ten

milliliters (mL) of peritoneal fluid should be sent for culture. Candida species usually grow quickly in culture but

other fungi may take weeks to grow.

When fungal yeast or hyphae forms are detected, systemic antifungal therapy should be started promptly. The

drug of choice will depend upon the organism that is cultured. Oral fluconazole 200mg/day for two to four weeks

is suggested for Candida albicans. In addition to antifungal therapy, the peritoneum should be lavaged until the

fluid is clear. It is usually recommended that the peritoneal catheter be removed and the patient be placed on

hemodialysis for four to six weeks before replacing the catheter and resuming peritoneal dialysis.

The morbidity and mortality of fungal peritonitis in these patients is greater than that seen with bacterial peritonitis.

Complications include sclerosing peritonitis, adhesions, bowel obstruction and stricture, fungal invasion of the

bowel wall, and abscess formation. Mortality rates are reported from 15 to 45 percent.

Prophylactic antifungal agents are recommended for peritoneal dialysis patients who receive prolonged

antibiotics. These patients are more prone to fungal peritonitis, which may be due to a shift in the balance of the

skin and bowel flora caused by antibiotics. Or, these patients may be at general risk for bacterial and fungal

infections due to poor peritoneal dialysis technique. Routine antifungal prophylaxis is not recommended for all

patients due to the risk of developing resistant organisms.

References:

1. Bryant JB, Corliss J, Crowley K, Elbaum DA, Long GJ (writers), and Martin KA and Villalba C (editors).

Patient information: Peritoneal dialysis (The basics). UpToDate, 2012.

2. Bryant JB, Corliss J, Crowley K, Elbaum DA, Long GJ (writers), and Martin KA and Villalba C (editors).

Patient information: Hemodialysis (The basics). UpToDate, 2012.

3. Burkart, JM. Diagnosis of peritonitis in peritoneal dialysis. UpToDate, 2012.

4. Cox, GM, Kauffman, CA, Schwab, SJ. Fungal peritonitis in continuous peritoneal dialysis. UpToDate,

2012

5. Karcher, DS and McPherson, RA. Cerebrospinal, Synovial, Serous Body Fluids, and Alternative

specimens., Chapter 29, in Henry’s Clinical Diagnosis and Management by Laboratory Methods, Twenty-

Second Edition, McPherson, RA and Pincus, MR, eds. Elsevier, China, 2011

Martha R. Clarke, MD


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Cell Identification

VB

F-0

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Participants


Macrophage containing abundant

small lipid vacuoles/droplets (Lipophage)

336 83.0 Educational



Squamous epithelial cell 4 1.0 Educational



3 0.7 Educational

Malignant cell 2 0.5 Educational

Macrophage containing abundant small lipid vacuoles/droplets

2 0.5 Educational


The cell identified by the arrow is a macrophage, as correctly identified by 94.6% of participants.

Macrophages are large cells (15 to 80µm) that arise from monocytes (12 to 20µm), which are bone-

marrow derived cells that circulate in the blood. As monocytes migrate into tissues, they evolve

morphologically and become tissue macrophages. Monocyte/macrophage morphology is quite variable,

ranging from a typical blood monocytes to the morphology of a typical macrophage. The cell in this

photomicrograph is that of a typical macrophage, displaying a round to oval nucleus, abundant amounts

of cytoplasm, and many cytoplasmic vacuoles. Macrophages often show evidence of active

phagocytosis. The ingested material may include other blood cells or bacteria, fungi, hemosiderin,

remnants of digested materials, and/or cytoplasmic vacuoles.

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VB

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Participants





Erythrocyte, nucleated 2 0.5 Educational


1 0.3 Educational

The cell identified by the arrow is a mature erythrocyte, as correctly identified by 96.8% of participants.

The morphology of erythrocytes (6.7 to 7.8 µm) in body fluids is similar to those present in the peripheral

blood. Erythrocytes may appear crenated in certain fluids, but that finding is not clinically significant.

Erythrocytes are not usually found in significant numbers in body fluid samples, and their presence could

reflect hemorrhage or traumatic contamination. They may also be seen in association with many disease

states, such as pancreatitis or malignancy, as in this particular case.

13

VB

F-1

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Participants





14 3.5 Educational


Germinal matrix cell 1 0.3 Educational

Ventricular lining cell (ependymal or choroid cell)

1 0.3 Educational

The cells identified by the arrows are malignant cells (nonhematopoietic), as correctly identified by 69.4%

of participants. A variety of neoplastic cells may be found in body fluids, and their appearance is

dependent upon the type of underlying malignancy. Virtually any neoplasm can invade the serous

cavities, resulting in malignant cells in fluid specimens. As in the present case, malignant

nonhematopoietic cells frequently form cohesive cell clusters, in contrast to hematopoietic neoplasms that

are most often seen as scattered single cells. Cytologic features of nonhematopoietic malignant cells on

cytocentrifuge preparations include: high nuclear-to-cytoplasmic ratio, increased cell and nuclear size,

irregularly shaped nuclei, atypical nuclear chromatin patterns, large nucleoli, and a tendency to form large

clusters, frequently with nuclear molding. Occasionally, cell clusters may recapitulate a structure, such as

pseudo-gland formation with adenocarcinoma. With malignant tumors, a distinct population of abnormal

cells separate from the hematopoietic cells is present.

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VB

F-1

1

Participants



Neutrophil/macrophage with phagocytized fungi

4 1.0 Educational

Neutrophil, immature (metamyelocyte, myelocyte, promyelocyte)

1 0.3 Educational

The cell identified by the arrow is a neutrophil, as correctly identified by 98.8% of participants. The

segmented or band neutrophil is typically easy to recognize and has an appearance similar to that seen

in peripheral blood. The segmented neutrophil (10 to 15 µm) has a low N:C ratio of ~1:3 and contains

pale pink cytoplasm with specific granules, condensed nuclear chromatin, and segmented or lobulated

nucleus (two to five lobes normally), In body fluids, however, they can often show eccentric nuclear lobes

or morphologic changes due to cytospin preparation or autolysis, including nuclear pyknosis and

fragmentation.

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VB

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Participants



Plasma cell 7 1.7 Educational




5 1.2 Educational


Lymphocyte 2 0.5 Educational

Plasma cell, abnormal 2 0.5 Educational

Synoviocyte (synovial lining cell) 1 0.3 Educational

The cell identified by the arrow is a mesothelial cell, as correctly identified by 91.8% of participants. The

mesothelial cell (20 to 50 μm) normally lines pleural, pericardial, and peritoneal surfaces. These cells can

be shed individually or in clusters. When found in pairs or clusters, mesothelial cells have articulated or

coupled cell borders with a discontinuous outer border (clear spaces or “windows”) between many of the

cells. The nucleus is round to oval in shape with a definitive nuclear membrane and regular contour.

Nuclear chromatin varies from dense to fine, but it is evenly distributed. Multiple nucleoli may occur and

the nuclei may overlap; however, the nuclei remain of approximately equal size and shape. One or more

nucleoli may be present. The nuclear-to-cytoplasmic ratio is low (less than 1:1), and the nucleus may be

central or eccentrically placed. The cytoplasm is light to dark blue and may have a grainy texture, typically

dense grainy basophilia or even a crystalline/ground glass appearance to the perinuclear area. In

chronic effusions or during inflammatory processes, mesothelial cells proliferate and become very large.

The nuclear chromatin is less condensed and nucleoli may be prominent; however, the nucleus still

retains a definitive, smooth, nuclear membrane. Mesothelial cells can be phagocytic and resemble

macrophages, resulting in forms that have a morphology intermediate between mesothelial cells and

macrophages.

Ria Vergara-Lluri, MD

Joan Etzell, MD


16

Case Discussion for VBF-07 – VBF-12

This case illustrates a malignant peritoneal fluid accumulation (ascites) in a woman with ovarian cancer. Ovarian

cancer is the second most common gynecologic malignancy in the United States. It is the fifth leading cause of

cancer death in all women, with 21,880 new cases and 13,850 cancer-related deaths from ovarian cancer in the

United States in 2010. Ovarian cancer ranks fifth in cancer incidence in women by site, superseded by breast,

colon, rectum, lung, and uterine cancers. The lifetime risk of ovarian cancer in the general population of women is

1.4 percent. Frequently, the clinical symptoms of ovarian cancer are vague and nonspecific, and often include

pelvic discomfort or pain, abdominal fullness or bloating. The abdominal fullness may be secondary to the

pathologic accumulation of peritoneal fluid (which is also referred to as ascitic fluid).

Virtually any tumor can cause body fluid accumulation, but carcinomas of the lung, breast, ovary, and GI tract are

the most common. In the peritoneal cavity, the most common cause of malignant effusions is ovarian cancer.

However, ovarian tumors in body fluids can exhibit very similar morphologic appearance to tumors arising from

other sites (such as carcinomas of the breast, stomach, and pancreas, and even peritoneal mesothelioma). Thus,

an extensive clinical and surgical pathology work-up, often including a variety of immunohistochemical stains, is

required to definitively characterize the site of origin and differentiation of the tumor. In addition, the majority of

ovarian tumors are benign (e.g. serous and mucinous cystadenomas). Thus, the main role of the clinical

laboratory is in recognizing the presence of malignant cells in body fluids.

Malignant tumors share key morphologic characteristics that usually allow distinction from benign cells. However,

the distinction between reactive mesothelial cells and tumor cells with low to moderate cytologic atypia can

sometimes be quite challenging. It is important to note that no one morphologic feature is pathognomonic for

malignancy, and it is the constellation of findings that allow for identification of malignant cells. When considering

whether cells are benign or malignant, one should look at the architecture of the cells, cell size, the nuclear

details, and the cytoplasmic quality.

Non-hematopoietic tumors typically are comprised of large cells that demonstrate cell-to-cell cohesion, which

manifests as three-dimensional cell clusters in body fluids. Non-hematopoietic malignant cells (see Images 1 and

2) are usually large, tend to have a higher nuclear to cytoplasmic ratio than benign/reactive cells and often

demonstrate irregular nuclear contours and/or nuclear hyperchromasia, Large and abnormally shaped nucleoli

and/or atypical mitotic figures (e.g. tripolar mitotic forms) also support malignancy. Unusual cytoplasmic contents

in clearly malignant cells may also yield important diagnostic clues. For example, intracytoplasmic mucin almost

always indicates adenocarcinoma, melanin pigment may suggest a melanoma, and keratin could indicate a

squamous cell carcinoma.

17

Image 1: Peritoneal fluid with malignant cells, low power view. Note the tight clusters and groups of

malignant cells which are clearly enlarged in comparison to the background erythrocytes and inflammatory cells.

Image 2. Peritoneal fluid with malignant cells, high power view. The malignant cells are enlarged, in stark

contrast to the background polymorphonuclear cells (neutrophils) and the smaller and finely vacuolated

macrophages. In this example, the malignant cells display variable nuclear-to-cytoplasmic ratios, round to oval

nuclei, prominent nucleoli, and cytoplasmic vacuoles in a subset of cells.

In the clinical case presented for this challenge, the body fluid slide demonstrates many large, three-dimensional,

cohesive cell clusters, which are distinct from the background inflammatory cells and erythrocytes. Each cell

cluster contains large cells which display high nuclear-to-cytoplasmic ratios, with only a scant amount of

cytoplasm. Although the nuclear outlines are relatively smooth and regular, the nuclei demonstrate

hyperchromasia. In addition, some of the cells show large intracytoplasmic vacuoles. Taken all together, the

combined morphologic features cement the diagnosis of a malignant effusion, in this case, that which arose from

an ovarian carcinoma.

18

To arrive at a specific diagnosis of gynecologic cancer, the physician must perform a comprehensive clinical,

radiologic, and laboratory evaluation, and enlist the diagnostic expertise of an anatomic pathologist. Obtaining

imaging studies is essential to locate the mass, estimate its size, and examine whether the mass is solid and/or

complex. A serum glycoprotein CA-125 is often obtained, which has good utility in approximately 80% of

advanced ovarian cancer cases and is most predictive of ovarian cancer in postmenopausal women. However,

this CA-125 monoclonal antibody blood test is not specific for ovarian cancer, as it may be elevated in association

with benign gynecological conditions, such as endometriosis, pelvic inflammatory disease, pregnancy,

leiomyomas, etc. Based on the clinical and radiologic evaluation, a gynecologic oncologic surgeon may choose to

perform surgery and send peritoneal washing fluid to the cytopathology and clinical laboratories for analysis. At

the request of the surgeon, a frozen section may also be performed by a surgical pathologist, which can aid in the

subsequent intraoperative management of the patient. With comprehensive gross and microscopic evaluation, the

anatomic pathologist can thus assign the tumor to prognostic categories of benign, intermediate (known as

borderline tumors or tumors of low malignant potential), and malignant, as well as assign the tumor differentiation

to epithelial, germ cell, or sex-cord stromal type.

Thus, the most common role of the clinical laboratory is in the accurate morphologic assessment of involvement

by malignant cells in the peritoneal body fluid, which is important for initial cancer staging and in identifying tumor

recurrence. The finding of malignant cells in the peritoneal fluid upstages the patient to either a stage IC (if tumor

is limited to ovaries) or IIC (if tumor involves both ovaries with pelvic extension) based on AJCC staging

(American Joint Committee on Cancer, 7th edition). If the tumor shows microscopic evidence of peritoneal

metastasis or lymph node metastasis, the tumor is considered stage III. The prognosis is most favorable in early

(e.g. stage I-II) disease. Many ovarian cancers are discovered at a later stage, when they have spread outside of

the pelvis and/or show lymph node metastases. The treatment of ovarian cancer typically includes surgery and

chemotherapy, and in patients with advanced ovarian cancer, chemotherapy is initiated prior to surgery to

facilitate surgical resection. Prognosis generally depends on the clinical stage as well as the type of tumor

present. The patient in this clinical challenge presented with abdominal distension and weight loss as her initial

symptoms of ovarian cancer, and the presence of the malignant effusion supported a stage IC ovarian carcinoma.

In summary, this case exemplifies many of the morphologic findings that can be seen with a malignant peritoneal

effusion from ovarian carcinoma.

References:

1. Jemal A, Bray F, et al. Cancer statistics, 2010. CA Cancer J Clin 2010; 60: 277.

2. Sears D, Hajdu I. The cytologic diagnosis of malignant neoplasms in pleural and peritoneal effusions.

Acta Cytologica 1987;31:85-97.

3. Galagan KA, Blomberg D, Cornbleet PJ, Glassy EF, eds. Miscellaneous Cells and Criteria for

Identification of Malignant Cells in Wright-Giemsa Stained Cytocentrifuge Preparations, In Color Atlas of

Body Fluids: An Illustrated Field Guide Based on Proficiency Testing. Northfield, IL: College of American

Pathologists; 2006.

4. Zaloudek CF. Tumors of the female genital tract: ovary, fallopian tube, and broad and round ligaments. In

Diagnostic Histopathology of Tumors, volume 1, 3rd

edition. Fletcher CDM, ed. Churchill Livingstone

Elsevier: 2007.

5. DeMay, M. Practical Principles of Cytopathology. Chicago, IL: American Society of Clinical Pathology;

2007. American Joint Committee on Cancer, 7th edition.

Ria Vergara-Lluri, MD

Joan Etzell, MD,


“candida albicans” and “ovarian cancer” · candida albicans is an endogenous commensal...

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