Pleural fluid cytopathology

How to prepare a proper specimen for pleural fluid cytology & cytopathological analysis.

Pleural Fluid Cytopathology

Pleural fluid analysis is more than a typical ‘rounds’ question for interns and students.  This fluid contains important indicators of disease status.  Who among us hasn’t memorized pH levels, glucose and protein values?  (For a discussion on transudate versus exudate effusions, see the Medscape article by Jeffrey Rubins below.)

While pleural fluid analysis can be used to assist in the differential diagnosis of multiple conditions; pleural fluid cytopathology is often ordered when a more sinister condition like metastatic cancer is suspected.  Therefore, it is especially important for clinicians to ensure that pleural fluid cytopathology samples are collected, and sent in the most efficient and effective manner possible.  While there are few written guidelines regarding this process, here are some helpful tips based on interviews with several pathologists and the available literature.

Biopsy is best but fluid analysis is still helpful

While the gold standard for diagnosis is always tissue biopsy (in this case pleural tissue biopsy), this does not mean that cytopathological analysis is completely unnecessary.  In many cases, this fluid analysis gives a first look that aids in the diagnosis and staging of disease.  It is particularly useful for patients undergoing thoracentesis procedures, particularly when thoracentesis is performed in lieu of a more invasive procedure such as VATS (which allows for direct tissue biopsy.)

But do I still need to do a biopsy if the fluid analysis is negative?

However, there is often a catch-22 in the use of pleural fluid pathology which can lead to some confusion among patients and providers.  This catch-22 is related to the sometimes variable reliability of pleural fluid cytopathology for diagnosis of malignancy.  This means that the results aren’t always accurate.  As anyone in thoracic surgery can tell you, there have been numerous times when the fluid analysis results are reported as negative (for malignancy) even when the surgeon is (literally) staring at a pleural tumor in the operating room.  This means that a negative pleural fluid cytopathology result can not be used to rule out malignancy.

However, when the fluid is positive, it may save the patient from an additional procedure*.

Cytology versus cytopathology

“Cytology” is the generic term for the study of cells.  Cytopathology is the actual pathological investigation of free cells and tissue fragments, often for the diagnosis or treatment of cancer.

When used clinically, cytopathology is often used to distinguish between other more basic studies of bodily fluids or tissues.  This in-depth cellular examination is more critical in many cases than basic pleural fluid analysis.  This examination may include identification of immunological factors and tumor markers.  This is one of the tests that clinicians use to try and answer the question,“Is it cancer?”.  However, the answer is not always as straight forward.

Reliability and Predictive Value

This question is difficult to answer due to sometimes variable prognostic value of the fluid itself.  Even under the best of circumstances, reliability of this test (like most diagnostics) is less than 100%.  Different studies calculate the accuracy of pleural fluid analysis at detecting cancer vary wildly;anywhere from 10 to 80% has been reported in the literature with false negatives as the most frequent error (when discussing sensitivity and specificity). However, poorly prepared specimens may contribute to false results as well.

Ensuring optimal results by obtaining proper specimens

 Over the years, during different discussions with multiple pathologists as well as laboratory technicians, a common theme has emerged regarding the use of pleural fluid for pathology analysis.  Several of these individuals remarked that obtaining an accurate diagnosis was often difficult due to improper or suboptimal preparation of the pleural fluid, in addition to characteristics of the fluid itself.  What constitutes a ‘proper’ or ‘optimal’ pleural fluid is still (among lab technicians and pathologists) up to debate, but here are some general guidelines:

1. Send it all.

Due to the nature of pathology analysis which replies of the presence and identification of malignant cells within the fluid itself, a larger fluid specimen provides for a better sample.  When thoracentesis/ VATS or other drainage is being performed, and this yields 2 liters of fluid – send all two liters.  Don’t select out the first 25ml in a urine specimen cup, send it all.

There are no set guidelines for the amount of fluid necessary for cytopathology analysis. While malignancies have been successfully detected in amounts as small as 4ml, the rationale behind providing larger samples has been explained as ‘increasingly the likelihood of detecting the presence of cells indicative of malignancy’.

While the amount of fluid needed is currently up for debate among pathologists, sending too little fluid may result in a missed diagnosis, whereas an overabundance of fluid is more of an inconvenience to lab technicians.

Be sure to include the last frothy bits, which often contain more sediment/ cellular material than fluid recovered at the beginning of the sample.  (The content of this fluid may even vary due to the patient’s position – which is another reason to take a larger sample.) In a conscious patient, this may mean several minutes of discomfort, but encourage patients to take deep breaths, and cough so that as much fluid as possible can be removed.  (In patients with very large effusions, this may be a lengthy process as ‘short breaks’ are taken during the procedure to accommodate for fluid shifts.  This brings us to # 2.

2.  Keep it fresh: Talk to the lab about whether you should consider adding an additive like heparin or EDTA to your sample at the time of collection to prevent the degradation of cells.  Depending on when / where your sample is collected and sent – there may be significant delays in the processing of the collected sample.  Many pathologists report that after 4 hours there are significant changes in untreated pleural fluid kept at room temperature.

Consider this as you gather your sample;

–          Did you leave it in the patient’s room for the nursing staff to deliver?

–          Is it possible it may sit for several hours before arriving to the lab?

–          Is the lab well-staffed or will the fluid sit waiting for analysis by overworked, and stressed employees at a lab that may be inundated with many more urgent requests?

Guzman et al. (1992) and other researchers found that with the addition of EDTA to pleural fluid specimens, tumor cells were easily identified even after four days of storage.

Even if your facility doesn’t provide EDTA for your specimens, it’s a good bet that sending a syringe full of fluid from the bottom of a week-old pleurovac is probably not your best bet.

3.  Eliminate errors: Don’t make them guess!

Always personally label fluid and tissue samples completely with the patient’s name, reference number (as used by your institution), body site (ie. Right pleural space) as well as the ordering clinician’s name.  Include your phone number if you want to be called with the results or questions.

On the actual order, or lab requisition, provide additional information including patient symptoms, and pertinent history (ie. 63 year old with 40+ pk years of smoking, and history of asbestos exposure in Navy shipyards, now presents with pleural effusion, chest pain and 25 pound weight loss.)  Provide any special instructions as needed.   This allows the pathologist examining the patient’s specimens to correlate clinical history, symptoms and other available diagnostics with cellular findings and stains.

4.  Now do it again.  If the patient develops a second pleural effusion, go ahead and send that fluid too – particularly if the first sample was non-diagnostic.

*Depending on the patient’s clinical status/ symptomology.  As mentioned in a previous post, many patients with malignant pleural effusions may undergo additional procedures at some point in time for palliation of symptoms.


 American Society of Cytopathology – a great resource for interested readers.  The website also contains a ‘virtual slide atlas’ which includes case studies and several slides showing pleural fluid cytopathology. Click here for the case study of a 60 year old with pleural effusion.

Antonangelo L, Capelozzi VL. (2006). Collection and preservation of the pleural fluid and pleural biopsy. J Bras Pneumol. 2006;32 Suppl 4:S163-9. Portuguese.  These Brazilian authors from the University of Sao Paulo discuss the proper collection of pleural fluid specimens.  In this article, the authors make recommendations for the collection, storage and examination of pleural fluid for a variety of laboratory and microscopic tests.

Brandstetter RD, Velazquez V, Viejo C, Karetzky M. (1994). Postural changes in pleural fluid constituents. Chest. 1994 May;105(5):1458-61.

Guzman J, Arbogast S, Bross KJ, Finke R, Costabel U (1992).  Effect of storage time of pleural effusions on immunocytochemical cell surface analysis of tumor cells. Anal Quant Cytol Histol. 1992 Jun;14(3):203-9. No free full text available.

Porcel JM.  (2011).  Pearls and myths in pleural fluid analysis. Respirology. 2011 Jan;16(1):44-52.  Porcel advocates for smaller volumes, but an ‘inadequate sample’ should never be a reason for a missed diagnosis.  He also advises the addition of an additive if there are any anticipated delays (4+hours) in specimen processing.

Salyer WR, Eggleston JC, Erozan YS. (1975).  Efficacy of pleural needle biopsy and pleural fluid cytopathology in the diagnosis of malignant neoplasm involving the pleura. Chest 1975 May, (5) 536-9.  Classic article on the predictive value of pleural fluid cytopathology. A  pdf of Salyer et al is available here.

Rubins, J. (2013).  Pleural effusion workup.  From Medscape/  Pleural Effusion Workup pdf version.

Additional Resources

Shidham, V. B. & Falzon, M. (2010). Serous cavities.  Chapter 3 in  Diagnostic Cytopathology: Expert Consult: Online and Print (2010).  Grey & Kocjan (Eds).   Elsevier Health Sciences.