Flow cytometry, molecular analysis, and other special techniques (in Serous Fluid Cytopathology)

FLOW CYTOMETRY

Serous effusion is an appropriate specimen for flow cytometric analysis to confirm the diagnosis of malignant lymphoid neoplasm. However, unlike the role of flow cytometry in the diagnosis of lymphoid lesions by fine-needle aspiration or biopsies, which is well-accepted, its use in serous fluids is not largely utilized. However, recently its use in body fluid analysis has increased significantly. • It is usually difficult to differentiate between reactive lymphocytosis and malignant lymphoma in serous effusions, especially in low-grade malignant lymphomas, on morphology alone. Therefore, flow cytometric analysis can be useful in this situation.^[1]

FLOW CYTOMETRY AND HEMATOPOIETIC NEOPLASMS

When the diagnosis of lymphoma/leukemia is suspected on cytopathologic examination and/or the patient has had a previous history of lymphoma/leukemia, flow cytometry can be helpful in the diagnosis and the classification of these tumors. • The algorithms in Figures 1 and 2 and Table 1 summarize the approaches of using flow cytometry data for aiding in the diagnosis of hematopoietic lesions.

Close

Figure 1:

Algorithm for evaluation of serous effusion suspicious for lymphoma.

Export to PPT

Close

Figure 2:

Flow cytometry immunophenotyping of lymphomas/lymphoid leukemias.

Export to PPT

The main differential diagnoses when one encounters small lymphocytes are usually between low-grade lymphomas and reactive effusion. Flow cytometry is usually the most helpful in this setting to distinguish the two. The encountering of large neoplastic cells usually brings up the diagnostic consideration of blue cell tumors and other large cell tumors such as poorly differentiated carcinomas, melanomas, and sarcomas.^[2–4] Flow cytometric analysis is often less helpful in this circumstance (because the large lymphoma/blasts cells can be missed during sample processing). In such cases immunocytochemistry on cell-block sections is more useful [see Figures 1, 2 and Table 1].

UNIQUENESS OF FLOW CYTOMETRIC STUDIES OF HEMATOPOIETIC NEOPLASMS IN SEROUS EFFUSIONS

FLOW CYTOMETRY AND NON-HEMATOPOIETIC NEOPLASM

Unlike the flow cytometry role in hematologic neoplasm, the application of these techniques in non-hematopoietic neoplasm is not well accepted in clinical practice and is still under investigation. There have been studies using flow cytometry on non-hematopoietic neoplasms on bench fine needle aspiration specimens^[9] and paraffin-embedded tissue.^[10] However, such applications on serous effusions have not been reported in the literature.

The percentage of CD16+, CD56+, and CD3– natural killer lymphocytes by flow cytometry may be helpful in identifying metastatic adenocarcinoma in serous effusion.^[11,12] An association of a higher percentage of natural killer (NK) cells (12–33%^[12] and 29–68%^[11]) with metastatic adenocarcinoma has been reported. In contrast to this, mesotheliomas, lymphomas, leukemias, malignant melanomas, and reactive mesothelial hyperplasia showed a lower percentage of NK cells (1–16%^[12] and 2–20%^[11]). • It was further suggested that the presence of ≥12% NK cells should make the cytopathologist suspect the presence of metastatic adenocarcinoma.^[12]

ELECTRON MICROSCOPY

With an increasing use of immunohistochemistry, there has been a decreasing use of electron microscopy in the diagnosis of malignant neoplasm. The main usefulness of electron microscopy in serous effusion fluid is in the differential diagnosis between malignant mesothelioma and metastatic adenocarcinoma after cytopathologic confirmation of malignancy. The presence of slender and elongated microvilli, abundant intermediate filaments, and lack of secretory granules favored mesothelioma, whereas short stubby microvilli and secretory granules suggested a diagnosis of adenocarcinoma.^[13,14] As a result, overlapping features may exist and cause difficulty in interpretation.^[13] • The main challenge with serous fluid specimens as compared to tissue biopsy is to locate and identify the malignant cell unequivocally for ultrastructural evaluation. Spuriously, the reactive mesothelial cells in the background may be evaluated and misinterpreted as mesothelioma. Furthermore, in clinical practice, electron microscopic studies are considered expensive, laborious, lengthy, and, nowadays, the diagnosis is usually reached with the help of immunocytochemistry.

FLUORESCENCE IN-SITU HYBRIDIZATION (FISH) AND METASTATIC SEROUS EFFUSION

Application of fluorescence in-situ hybridization (FISH) technique has not become popular in serous effusion analysis. In hematopietic neoplasm, FISH analysis is standard practice to identify chromosomal structural and gene rearrangement abnormalities. A cell-block may be of more value for FISH testing than lose cells. In carcinoma, recent studies have demonstrated the possible role of this method for detecting malignant cells in effusions with higher sensitivity.^[15–22] The potential uses of FISH using different centromere specific probes on chromosomes 1, 3, 7, 8, 9, 10, 11, 12, 17, and 18 (with different combinations) have been examined in serous effusions for detecting pleural malignant mesothelioma,^[15] metastatic breast cancer,^[16] and metastatic carcinoma.^[16–21] The studies suggested that FISH can be helpful in cytologically negative or ambiguous effusions and yielded highly sensitive and specific results when used together with cytologic examination.^[15–22] Diagnostic superiority was demonstrated in metastatic effusions from the breast, lung, pancreas, gynecologic, and gastrointestinal carcinomas.^[18]

Furthermore, cases with known primary tumors associated with abnormal FISH patterns would facilitate the appropriate choice of centromeric probes for detection of metastasis.^[20] However, the limitation of FISH has been observed in cases consisting of a small population of malignant cells hidden against a background of inflammatory or reactive cells.^[21]

• It should be highlighted that cytomorphologic examination is still the gold standard for tumor staging and is the only method accepted in the classical AJCC cancer staging.^[18,23] Prospective studies are needed to demonstrate the clinical benefit of FISH to detect disseminated tumor cells in correlation with clinical outcomes.^[18]

MOLECULAR GENETICS

MOLECULAR GENETICS AND SOFT TISSUE TUMORS

The knowledge of molecular pathogenesis of soft tissue tumors has grown tremendously over the last decade. Molecular diagnostic techniques, particularly reverse transcriptasePCR (RT-PCR) and FISH, have become important tools to detect the characteristic fusion genes associated with soft tissue tumors [Table 4]. Nonetheless, the application of these techniques to serous effusion specimens is rarely required in clinical usage for two reasons. First, sarcomas rarely cause serous effusions. Secondly, when sarcomas are present in serous effusions, the diagnosis of sarcoma has almost always already been established^[2,37] Table 4 summarizes the major molecular genetic abnormalities in soft tissue tumors.

POLYMERASE CHAIN REACTION FOR DIAGNOSIS OF PLEURAL TUBERCULOSIS

Although the detection of mycobacterial DNA in pleural effusions by PCR appears to be a promising approach for the rapid diagnosis of pleural tuberculosis, there is no consensus regarding the usefulness of PCR testing for the diagnosis of pleural tuberculosis.^[38] The reported sensitivity of PCR testing for pleural tuberculosis varies, ranging from 17.5 to 83%,^[38–47] probably due to different PCR methods and diverse clinical diagnostic criteria. The reported specificity of the tests ranged from 78 to 100%, approaching 100% in most.^[38–47] The above data suggest that PCR is not recommended in a routine clinical practice, although clinical utility of PCR testing for diagnosis of pleural tuberculosis may be improved in the future.

OTHER TECHNIQUES

In this section, we describe techniques that are applicable to serous effusions, but their role at present in routine clinical practice is controversial. For a detailed review of the emerging techniques for serous effusion application, please refer to other timely articles such as that by Ross.^[48]

CYTOGENETICS

Although effusion fluid is suitable for cytogenetic evaluation, the use of this technique in the clinical specimen is limited. Several reports suggested the role of cytogenetics in distinguishing malignant and benign pleural effusions by using the presence of several chromosomal aberrations in neoplastic effusions.^[49] It was initially believed that aneuploidy or polyploidy were specific for malignant cells, with the specificity approaching 100% in the diagnosis of malignant effusions. However, benign reactive mesothelial cells with chromosomal abnormalities have been described.^[49] Moreover, the technique itself also limits its application to clinical specimens. It is laborious, time consuming, and requires fresh material, with a high number of malignant cells, specialized laboratory facilities, and trained technologists and cytotechnologists.

CHROMOGENIC IN-SITU HYBRIDIZATION (CISH)

CISH is a non-fluorescent alternative to FISH. The technique is feasible in the clinical laboratory and with routine specimens. The application to fine-needle aspirates of breast cancer has been described.^[50] Nonetheless, such applications to serous effusions have not been reported extensively in the literature.

DIGITIZED IMAGING

A study describing the possible usefulness of digitized imaging in serous effusions has been reported.^[51] However, more studies need to be published to evaluate the interobserver variability to confirm its clinical applications.

PROTEOMICS; TWO-DIMENSIONAL GEL ELECTROPHORESIS; MALDI AND SELDI

Since the potential use of high-throughput mass-spectroscopy based blood for the detection of localized ovarian cancer by Petricoin et al.^[52] was published, a few studies using the same method for detection of metastatic carcinoma in serous effusions have been published.^[53,54] The methods are expensive and at present are mostly applicable as research tools.

DNA PLOIDY ANALYSIS

Although the detection of DNA aneuploidy by using flow cytometric methods can be performed in the clinical laboratory and is economical, this method has not been implemented as a routine diagnostic screening tool in serous effusions. The literature data suggests a high false-negative rate.^[5]

CASE STUDIES

Case 1

History

A 78-year-old female presented with dyspnea. A chest X-ray revealed pulmonary vascular congestion, pulmonary edema, and bilateral pleural effusions. The heart was markedly enlarged with globular configuration, suggestive of pericardial effusion. A pericardiocentesis was performed and submitted for cytologic examination and cell-surface flow cytometric analysis.

Cytomorphologic evaluation

The Giemsa and Papanicolaou (PAP)-stained Cytospin preparations [Figure 3a,b] showed a monomorphic population of medium to large lymphocytes admixed with a few acute inflammatory cells.

Close

Figure 3:

Diffuse large B-cell lymphoma, pericardial fluid. (a,b) A predominantly monotonous population of medium-to-large lymphoid cells (arrows) admixed with a small number of acute and chronic inflammatory cells. [a,b, 40X (a, Giemsa-stained Cytospin preparation; b, PAP-stained Cytospin preparation)] (c–h) The two-dimensional histogram of four-color flow cytometric evaluation. The two-dimensional histograms show four-color flow cytometric analysis of B lymphocytes, which express CD19 and CD20 (d–f). These neoplastic B cells are medium to large, as illustrated by intermediate forward scatter (FSC) (c), correlating with the cytomorphologic correlation (a,b). They also co-express CD10 (e,f), are negative for CD5 (h), and demonstrate kappa light chain restriction (g).

Export to PPT

Provisional cytomorphologic interpretation

Suspicious for lymphoma. Final interpretation pending immunophenotyping (flow cytometric analysis).

Flow cytometric findings

• Four-color flow cytometric analysis of B lymphocytes (depicted in red) showed expression of CD19 and CD20 [Figure 3d-f]. These neoplastic B cells are medium to large, as illustrated by intermediate forward scatter (FSC) [Figure 3c], correlating with the cytomorphologic findings [Figure 3a,b]. They also co-express CD10 [Figure 3e,f] with kappa restriction [Figure 3g] and with negativity for CD5 [Figure 3h].

Final diagnosis

Large B-cell lymphoma involving pericardial cavity.

Cytomorphologic evaluation

Giemsa and PAP-stained Cytospin smears [Figure 4a,b] were hypocellular and showed small mature lymphocytes resembling chronic inflammatory cells admixed with polymorphonuclear cells and reactive mesothelial cells.

Close

Figure 4:

Follicular lymphoma, ascitic fluid. (a,b) Mixed hypocellular population of small benign-appearing lymphocytes (L), occasional polymorphonuclear cells (N) and reactive mesothelial cells (RM). [a,b 40X (a, Giemsa-stained Cytospin preparation; b, PAP-stained Cytospin preparation)] (c–h) The two-dimensional histogram of four-color flow cytometric evaluation. The two-dimensional histograms show four-color flow cytometric analysis of B lymphocytes, which express CD19 and CD20 (d–f). These neoplastic B cells are small to medium, as illustrated by intermediate forward scatter (FSC) (c), correlating with the cytomorphologic correlation (a,b). They also co-express CD10 (f), are negative for CD5 (h), and demonstrate kappa light chain restriction (g).

Export to PPT