mgh:cyto-1-5

• Ivan Chebib MD, Amy Ly MD, Ron Arpin SCT
• Reading: Cibas 4th Ed. Chapter 6; Bibbo/Wilbur 4th Ed. Chapter 16
• Questions: When you have completed unit 1-5 go to the Assessment tab and answer the question.
• Dr. Tambouret CSF Lecture

• Indications for cytology examination

by Amy Ly, M.D.
The pleural, pericardial, and peritoneal cavities are lined by serosa, which is a simple layer of mesothelial cells. Under normal conditions, these cavities contain only a small amount of fluid which allows adjacent serosal surfaces to move over each other with low resistance during normal organ activities (e.g. breathing, heartbeats, peristalsis). In disease states, a greater amount of fluid accumulates and is called an effusion. Effusions may be characterized clinically as transudative or exudative. Transudates result from unbalanced hydrostatic and oncotic pressures. Exudates result from injury to the mesothelium, which is commonly caused by malignant tumors that have spread to serosal surfaces and/or malignant mesothelioma that originates in the serosa.

Detection of serosal malignancy by cytologic exam is more sensitive than by blind biopsy (58%-71% compared with 45%). Cytology sensitivity is further increased by 2%-38% if more than one sample is examined. However, the false negative rate is still significant. If cytology is negative but there is high suspicion for pleural malignancy, the patient can undergo thoracoscopy for further evaluation.

The specificity of cytologic effusion evaluation is very high: the false positive rate is <1%. False positive and false suspicious diagnoses are mainly due to reactive mesothelial cells that appear atypical.

Gynecologic and non-gynecologic malignancies involving the peritoneal serosal surfaces may not produce an effusion or be associated with lesions visible by gross inspection intraoperatively. In such cases, the peritoneal cavity may be evaluated by “peritoneal washing,” which is part of a cancer staging procedure. Peritoneal washings may also be used to exclude occult malignancy in patients undergoing laparoscopy or laparotomy for presumed benign gynecologic conditions and in women with BRCA1/2 mutations undergoing risk reducing salpingo-oophorectomy. Peritoneal washing may be potentially utilized to monitor a patient's response to adjuvant treatment for cancer.

Peritoneal washings that are positive for malignancy are associated with worse prognosis in patients with ovarian and fallopian tube cancers. Positive washings alone impact the surgical stage of only 3-5% of women with gynecologic cancers, but may be the only evidence of metastasis to the peritoneum for some patients. Peritoneal washing results are included in the International Federation of Gynecology and Obstetrics ovarian and fallopian tube cancer staging algorithm. The prognostic utility of this test for endometrial and other gynecologic cancers is unclear at this time.

There is a significant false-negative rate with peritoneal washings. 23-86% of patients with biopsy proven peritoneal metastasis have no evidence of disease in their washings by cytologic exam. The high false negative rate may be partly due to poor distribution of fluid within peritoneal cavities that have been affected by adhesions. False positive diagnoses are not common (<5% of cases), and are usually due to proliferative mesothelial cells with reactive changes and associated psammoma bodies, and endometriosis.

• Procuring the specimen

by Amy Ly, M.D.
Effusion specimens are obtained by inserting a needle into the pleural space (thoracentesis), pericardial space (pericardiocentesis), and peritoneal cavity (abdominal paracentesis). Peritoneal fluid is usually obtained through the abdominal wall, however in women it may also be aspirated from the cul-de-sac through the vagina (cold to centesis). Effusions may also be collected during thoracic, abdominal, or cardiac surgery. Removing this excess fluid may be performed for diagnostic purposes (submitted for pathology evaluation) or therapeutic purposes (to alleviate symptoms such as dyspnea and heart failure). Large volumes (several liters or more) of abdominal fluid may be drained safely. However, pleural fluid that is rapidly removed in large quantities may rarely be complicated by reexpansion pulmonary edema. This condition is fatal in up to 20% of cases and tends to involve younger patients with a long duration of lung collapse who experience rapid lung reexpansion upon thoracentesis.

The effusion is collected in sterile containers and sent unfixed to the laboratory. Specimen collection into glass containers causes rapid clotting, which is undesirable as this causes dispersion of cells and makes it more difficult to evaluate them. To prevent clotting, collect fluids into heparinized bottles containing 3 units of heparin per milliliter of capacity. If heparinized bottles are not available, the heparin should be placed into the container before the fluid is drained. Store fluids at 4°C until the time of slide preparation. Effusions are robust specimens and may be refrigerated for > 2 weeks without compromising cellular morphology or antigenicity for immunostains because the effusion itself nourishes the cells within it. However, specimens involved by malignancies with high cellular turnover (e.g. Burkitt lymphoma) should be prepared as soon as possible.

Peritoneal washes are obtained intraoperatively. The surgeon evacuates any pre-existing peritoneal fluid and submits it separately for cytologic examination. Sterile saline (50-200 mL) is instilled into multiple areas, usually the pelvis, the right and left paracolic gutters, and the undersurface of the diaphragm. A repeat washing or rinsing action is used to abrade cells from the serosal surfaces into the saline. The saline is then pooled into a single collection and heparinized. There is no advantage to submitting washings from different sites separately. The specimen should be delivered to the laboratory unfixed and stored at 4°C until slides can be prepared. If there will be a significant delay before slide preparation, an equal volume of 50% ethanol can be added to preserve the specimen.

• Test platforms/specimen processing and triage

by Amy Ly, M.D.
To make slides from an effusion, the first steps are to agitate the fluid to evenly disperse the cells and then to centrifuge up to 50 mL of the fluid. The supernatant is discarded and the pellet is used to prepare smears, cytocentrifuge preparations (Cytospins), or thin-layer preparations (e.g. ThinPrep, SurePath). The slides are usually alcohol fixed but if a lymphoproliferative disorder is suspected, air dried Cytospins are helpful. Slides are stained with a Papanicolaou or Romanowsky type stain. Residual fluid is set aside in case additional slides or other preparations/tests such as cell block, flow cytometry, and molecular studies are needed.

Cell blocks may be prepared from fluids by coagulating the sediment into a compact mass with plasma and thrombin, wrapping the sediment in filter paper, placing in a cassette, and processed in the manner of histologic sections (fixing in formalin, embedding in paraffin, cutting, and staining with H&E). Clots that are already present in the fluid because it was not heparinized should be placed in cassettes for processing as cell blocks. The addition of a cell block to a smear/Cytospin/thin-layer slide increases sensitivity for the detection of malignancy. Cell block sections are useful for special and immunohistochemical stains. Cell block sections are also convenient for morphologic comparison with histologic sections because the tissues have been processed in an identical manner.

To prepare slides from a peritoneal washing, the specimen is thoroughly mixed and 50 mL of fluid is centrifuged. The supernatant is discarded and the pellet can be used to prepare smears, cytocentrifuge preparations (Cytospins), or thin-layer preparations (e.g. ThinPrep, SurePath). The remaining material or a separately centrifuged cell pellet can also be fixed in 10% formalin and processed as a cell block, employing histologic methods of processing, paraffin embedding, cutting, and H&E staining. Cell block sections are useful for morphologic comparison to the patient's resected neoplasm and for performing special and immunohistochemical stains.

• Reporting and terminology

by Amy Ly, M.D.
There are no established criteria for adequacy of effusion specimens. Cytologic diagnosis of fluids utilizes the following categories: “no malignant cells identified,” “atypical” (low suspicion for malignancy), “suspicious” (high suspicion for malignancy), and “positive for malignant cells.” The diagnosis of malignancy is semi-quantitative and semi-qualitative. “No malignant cells identified” and “positive for malignancy cells” are self-explanatory unequivocal diagnoses. Indeterminate categories of “atypical” and “suspicious for malignancy” are used when abnormal cells are present, but are too poorly preserved or too few in number to render a definitive diagnosis of malignancy. Approximately 5% of specimens are diagnosed as “suspicious.” In such cases, the effusion will usually re-accumulate if there is a serosal malignancy; the next sample may contain evidence of malignancy.

Adequacy criteria for peritoneal washing cytologic specimens have not been established, but there should be at least a few groups of well-preserved benign mesothelial cells present before concluding that the specimen is adequate for evaluation and negative for malignant cells. Specimens with malignant cells are always adequate. Results of peritoneal washing cytology are commonly reported as negative, atypical, suspicious, or positive for malignant cells. Atypical and suspicious interpretations should be avoided if possible because they are not helpful for treatment decision-making. Usually, only an unequivocally positive diagnosis is used for staging purposes, and atypical and suspicious results are considered to be negative results. Equivocal cytology washing cases may be resolved by comparing morphology a current corresponding resection specimen.

Introduction

CSF is produced by the choroid plexus in lateral, 3rd and 4th venticles by passive filtration and active transport. The CSF circulates through the subarachnoid space from the ventricles to bathe the brain and spinal cord. The Chorioid plexus consists of frond-like villous projections of vessels and pia mater that protrude into the ventricles. Specialized ependymal cells known as choroidal epithelium overlies the villi. The CSF is resorbed in the archanoid villi in the superior sagittal and intracranial venous sinuses and around spinal nerve roots. The arachnoid villi function as one way valves.

CSF Circulation

Indications for cytology examination

The CSF is examined in many clinical situations. The CSF is submitted for cytology examination usually only when a malignancy is suspected, either metastatic solid tumors or lymphoma/leukemia. Leptomeningeal (LM) metastasis is diagnosed in about 5% of patients with metastatic carcinoma. The tumors most likely to involve the CSF in order of frequency are breast, lung, melanoma, GI tumors and carcinoma of unknown primary. Primary brain tumors can involve the CSF. Forty percent of primary CNS lymphomas will have LM involvement

Accuracy

The sensitivity of CSF cytology for malignancy ranges from 80 to 95%. False-positive results are very rare, but false negative (FN) results are not uncommon. To minimize FN a minimum of 10 cc CSF should be sent to cytology, the sample should be processed promptly and a repeat sample obtained if malignancy suspected but results are negative. One study showed increasing sensitivity with repeat samples of 71% for first, 86% for second, 90% for third and 98% for > 3 samples (Glantz MJ et al. Cancer 1998;82:733).

Procuring the CSF sample

Usually the CSF is obtained by lumbar puncture. Samples may also be obtained from an Ommaya resevoir which consists of a subcutaneous pouch connected to a cannula ending in one of the lateral ventricles.

Test platforms/specimen processing and triage

Currently in the MGH lab two cytospin slides are prepared from a fresh CSF sample. One slide is fixed in 95% ethanol immediately after preparation and stained with Papanicolaou stain. The second is allowed to air dry and then is stained with rapid Giemsa stain used at MGH.

IMG_0737.MOV

The CSF Cytology Report

The results are report as one of four categories: Negative for malignant cells, Atypical (low degree of suspicion for malignancy), Suspicious (a high degree of suspicion for malignancy) or Positive for malignant cells. Over 90% of CSF samples are reported as Negative.

Basic cytomorphology

Normal CSF – N13-7784

• Rare lymphocytes, monocytes and PMN's
• Occasionally, ependymal cells, arachnoidal cells and choroid plexus cells are found
• Squamous cells, chondrocytes and red blood cells may be found as contaminants
  
  

Lymphoma – N13-7674

• Singly distributed, usually monomorphic population of cells with high N:C ratio
• Nuclei are irregular with clumped chromatin
• Macronucleoli may be present
• Mitotic activity may be evident
  
  

Melanoma –N11-14115

• Cells are usually singly distributed with occasional loose clusters
• Nuclei are round to oval, centrally or eccentrically located and may be multiple
• Nuclear chromatin is vesicular with eosinophilic macronucleoli
• Coarse, brown melanin granules may be present within the cytoplasm
  
  

Metastases – MN05-X04913

• Adenocarcinoma - cells are present singly or in small clusters
• Nuclei are irregular, three dimensional and eccentrically located
• Nucleoli are often present
• May be cytoplasmic vacuolization
• Small cell carcinoma - cells are present in small, molded groups
• Nuclei exhibit classic salt and pepper chromatin pattern and may be angular
• Cells have only a scant rim of fragile cytoplasm
  
  

---

Cytology service page