Fine needle aspiration biopsy and flow cytometry in the diagnosis of lymphoma.

Fine needle aspiration biopsy (FNAB) is emerging as a technique of potential value in the diagnosis of benign and malignant lesions in areas such as the breast and thyroid gland. Its place in distinguishing reactive from neoplastic lymphoid proliferations, when compared to the established practice of excision biopsy and histopathology, continues to undergo evaluation. Morphology alone discriminates poorly between atypical or lymphoproliferative disorders as seen in the presence of Epstein-Barr or human immunodeficiency virus. Furthermore the polymorphic populations of follicular lymphoma may mimic reactive changes. In addition previous classifications of these tumours using working formulation or Kiel classification relied heavily on architecture, which is a feature not reflected in cytology smears. The World Health Organisation approach includes clinical features, immunophenotyping and cytogenetic profiles to define neoplasms of immunohaematopoietic tissues. Flow cytometry on fine needle aspiration biopsy offers additional advantages in being rapid and objective in quantitatively as well as qualitatively documenting cell surface characteristics. All patients referred for this procedure to Tygerberg Academic Hospital with suspected nodal or extranodal lymphoma between January 2002 and December 2004 were analysed. In each case flow cytometry and cytomorphology were correlated with histopathology on tissue biopsy, bone marrow examination and clinical follow-up for confirmation of diagnosis. Results of the 124 cases were tabulated and statistically processed. Eighty-one met the inclusion criteria, thirteen (16.1%) were not malignant, two (2.5%) were falsely negative, two (2.5%) were equivocal needing histology and in the remaining sixty-four (79%) diagnosis was achieved. SUMMARY: Fine needle aspiration coupled with flow cytometry can reliably distinguish between nodal and extranodal neoplastic B-cell population. It is concluded that appropriate use, in a collaborative multidisciplinary setting, may eliminate the need for surgical procedures in many cases. CONCLUSION: These advances are not widely recognised and this is particularly true in South Africa. Accordingly, such an approach has been prospectively evaluated in the Western Cape showing that the combination of ready availability and diagnostic accuracy, after an initial learning curve, allow accurate characterisation of haematologic malignancies so that excision biopsy may be reserved for specific further studies to provide data not available from this less invasive procedure.     

AIDS-related lymphoma diagnosed by flow cytometry of a pleural effusion

We have presented a case in which flow cytometry of pleural fluid was applied in the diagnosis of an AIDS-related lymphoma. Flow cytometric evaluation of malignant effusions is a less invasive means of diagnosing these neoplasms than tissue biopsy.     

Quantitative flow cytometry.

Flow cytometers are instruments that can quantify fluorescence intensity data and provide unique information about cell populations. Significant advances have been made in terms of calibration reagents, evaluation of the quality and limitations of the monoclonal antibodies, standardized sample preparation, and data analysis to ensure interlaboratory comparability and reproducibility. Efforts to standardize quantitative fluorescence intensity measurements impact current clinical flow cytometry applications (i.e., immunophenotyping), but also emerging technologies, such as microarray assays, which also require calibration of fluorescence intensity across different platforms.     

HLA‐B27 testing: A journey from flow cytometry to molecular subtyping

Background

Determination of HLA‐B27 status plays an important role as adjuvant in suspected cases for diagnosis of Ankylosing Spondilytis (AS). Objectives of this study were to evaluate (i) flow cytometry method in comparison with DNA microarray for HLA‐B27 typing and (ii) EUROArray HLA‐B27 Direct assay for HLA‐B27 allele detection along with discrimination of AS/non‐AS subtypes in Indian population.

Methods

A total of 7543 patients with a presumptive clinical diagnosis of AS were referred for screening of HLA‐B27. All samples were initially tested by flow cytometry, and based on its findings, 1560 samples were analyzed for the presence of HLA‐B27 allele by microarray technology. A subset of samples (n = 200) were further tested by DNA sequencing for identification of HLA‐B27 subtypes.

Results

Screening of HLA‐B27 by flow cytometry reported 1551 positive (20.56%) and 5556 negative (73.65%) cases. Remaining 436 (5.78%) samples were identified within equivocal zone. Of cases (n = 1560) analyzed by microarray method, 1333 (85.44%) and 227 (14.55%) were detected microarray positive and negative, respectively. DNA sequencing identified HLA‐B*27:07 as the predominant subtype among cases showing ex2 positivity by microarray method. Of 200 cases, 20 cases (14 of HLA‐B*07 and 6 of HLA‐B*37) of HLA‐B27 cross‐reactive subtypes were also identified.

Conclusion

We recommend DNA typing as a complementary tool along with flow cytometry to accomplish successful HLA‐B27 phenotype determination. This is the first study among Indian population to evaluate efficacy of EUROArray to detect B27 allele and its potential to indicate the presence of nondisease‐associated alleles in Indian population.

     

Basics of flow cytometry.

The basics of flow cytometry are described. Cytometry is the measurement of cells; the flow cytometer performs this measurement quickly and reproducibly. Single cells in suspension are presented to a light source through hydrodynamic focusing. The resultant scattered light and fluorescence are collected and converted to electrical pulses, which are digitized for computer analysis. Frequency distributions can be viewed as single- or multiple-variable histograms for evaluating cell measurements. The versatility of flow cytometry is demonstrated by the types of samples it can analyze, characteristics it can measure, and applications in multiple laboratory sciences. Flow cytometry has been adapted easily and rapidly from a research tool to a clinically important procedure.     

用流式细胞仪分析骨髓细胞诊断淋巴瘤

FCM已经成为临床诊断淋巴瘤的有力工具.骨髓细胞FCM诊断的主要依据是SSC和CD_(45)细胞分布图上是否出现大小和分布不同于正常细胞的异常细胞,这种细胞是否存在和缺失相应的细胞表型.B细胞淋巴瘤的克隆性用免疫球蛋白к或λ轻链限制性确定,然后根据CD_5和CD_(10)表达与否,分为4种类型,进一步区分B细胞淋巴瘤亚型.T细胞淋巴瘤的克隆性用TCRα/β和γ/δ,TCR Vβ 25个家族的限制性确定,然后根据CD_4和CD_8表达与否,分为4种类型,进一步区分T细胞淋巴瘤亚型.NK细胞瘤可用CD_(56)、EBV、细胞毒颗粒TIA-1、颗粒酶B和穿孔素阳性与否进一步确定亚型.     

Clinical applications of flow cytometry.

OBJECTIVE: To describe the basics of three clinical applications of the flow cytometer. DATA SOURCES: Recent articles and books on flow cytometry and laboratory diagnosis. STUDY SELECTION: Not applicable. DATA EXTRACTION: Performed by the author. DATA SYNTHESIS: Immunophenotyping is the classification of cells based on antigens present on their surfaces. These antigens can be detected and quantified by a flow cytometer using monoclonal antibodies conjugated to fluorescent dyes. Reticulocytes are immature red blood cells that contain varying amounts of RNA and DNA. They can be stained with a fluorescent dye and enumerated by a flow cytometer. DNA ploidy analysis of solid tumors involves staining the nuclei of cells with a fluorescent dye. The amount of DNA in each cell is determined, and the percentage of cells in the S phase is calculated. CONCLUSION: Flow cytometry is a relatively new technology in the clinical laboratory. There are many clinically useful applications for which it is suited. As the technology continues to grow, so will the use of the flow cytometer.     

Oncology applications of flow cytometry.

The application of flow cytometry in evaluation of neoplasms is reviewed, with emphasis on DNA content analysis and surface-membrane antigen analysis. By measuring total nuclear DNA content one can determine the ploidy status and S-phase fraction of tumor cells. Aneuploidy, an abnormal DNA content, is a marker of malignancy and often a predictor of poor prognosis in a number of tumor categories. S-phase fraction reflects the proliferative activity of the tumor and is therefore related to the aggressiveness of the tumor. Surface-membrane antigen analysis of hematologic malignancies using monoclonal antibodies is useful in determining lineage and differentiation of tumor cells. This information is helpful in diagnosis and monitoring of patients and also has prognostic values. The flow cytometry is a new tool for the management of cancer patients because of its role in diagnosis, monitoring, and prognosis of tumors.     

Clinical applications of flow cytometry in hematology and immunology.

OBJECTIVE: To review the major applications of flow cytometry in the diagnosis of hematologic and immunologic disease. DATA SOURCES: Professional literature and authors' experience. DATA SYNTHESIS: Flow cytometry is evolving as a major diagnostic tool for evaluating hematologic disease, monitoring HIV infection and assessing peripheral blood and bone marrow for CD34 positive stem cells in marrow transplantation. This review highlights the opportunities and pitfalls of this area of diagnostic laboratory medicine. CONCLUSION: The applications of flow cytometers in diagnostic hematology and immunology are expanding, providing new opportunities for enhanced precision in diagnosis. As in any relatively new field much work remains in order to optimize accuracy and efficiency while minimizing cost.     

Transgastric endoscopic ultrasound-guided fine-needle aspiration biopsy and flow cytometry of suspected lymphoma of the spleen

BACKGROUND AND STUDY AIMS: Masses in the spleen can be sampled by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) but the diagnosis of lymphoma using EUS-FNA and flow cytometry has not been reported. We report our experience with transgastric EUS-FNA and flow cytometry in the investigation of patients with suspected lymphoma of the spleen. PATIENTS AND METHODS: All patients with splenic lesions that had been detected by computed tomography and who were referred for transgastric EUS-FNA over a 3-year period were enrolled in this study. The tissue obtained by EUS-FNA was evaluated by flow cytometry in all patients. RESULTS: Six patients with splenic masses were enrolled (four men, two women; median age 58.5 years, range 41 - 82 years). The mean size of the short axis of the lesions was 37.8 mm (SD 23.76 mm) and the mean size of the long axis was 45.6 mm (SD 31.72 mm). EUS-FNA was performed successfully in all patients and the tissue obtained was evaluated by flow cytometry. Two patients were diagnosed with lymphoma; no pathology was identified in the other four patients. Lymphoma of the spleen appeared as sharply demarcated echo-poor lesions; benign lesions appeared echo-rich in comparison with the surrounding splenic tissue. The two patients who were diagnosed with lymphoma underwent chemotherapy. Of the four patients in whom no pathology was identified, one patient subsequently underwent splenectomy for evaluation of persistent abdominal pain and was diagnosed with lymphoma; the three other patients had true-negative disease on the evidence of long-term follow-up (mean 8 months; range 6 - 12 months). No complications related to the EUS-FNA procedure were encountered in any patient. CONCLUSIONS: EUS-FNA of spleen masses is a safe technique that aids in the diagnosis of lymphoma when used in conjunction with flow cytometry.     

Urine flow cytometry and detection of glomerular hematuria.

BACKGROUND: The UF-100 is a flow cytometer designed for automated cellular urinalysis. In this study, the usefulness of the UF-100 in laboratory investigation into the origin of hematuria was evaluated. METHODS: Results from flow cytometric urinalysis were used to classify urinary red blood cells (RBCs) according to glomerular and non-glomerular origin and the classification was compared to the patient's clinical diagnosis as the gold standard. In parallel, microscopic sediment analysis was carried out. RESULTS: A total of 206 urine samples from 129 patients were analyzed (127 from patients with glomerular hematuria, 79 from patients with non-glomerular hematuria). Of these, 136 samples (92 patients) showed overt hematuria (>or=20 RBC/microL). Urine flow cytometry correctly classified 61% (sediment analysis 69%) of urine samples with overt hematuria. If inconclusive results are excluded, the UF-100 correctly diagnosed 85% (sediment analysis 98%) of urine samples with overt hematuria. The UF-100 and microscopic sediment analysis both showed sensitivity of 99% for the detection of glomerular hematuria. The specificity of the UF-100 for the detection of glomerular bleeding was lower (42%) than the specificity of microscopic sediment analysis (93%). CONCLUSIONS: Owing to its low specificity, the UF-100 showed limited capacity to discriminate glomerular from non-glomerular causes of hematuria in a population with a high incidence of renal disease. Therefore, extensive microscopic urinalysis remains necessary to assess the origin of hematuria.     

Clinically relevant functional flow cytometry assays.

As a lymphocyte proceeds along the pathway of cell division from the very earliest events of receptor aggregation-kinase activation to the final physical process of cell division, several physiologic functions occur, which can be measured by flow cytometry. Many of the functions along this pathway can be induced and measured in vitro and have led to the development of clinically relevant tests, which have been reviewed as functional flow cytometry procedures. It must be noted, however, that in addition to all of the physiologic processes that occur in lymphocytes (and in fact most cells) along the pathway to proliferation there are also several differentiated immune functions that are subject to flow cytometric analysis. Procedures for the evaluation of immune functions, such as phagocytosis, cellular aggregation, natural killer-cell cytotoxicity, cytokine secretion, antibody secretion, and antigen-specific cellular cytotoxicity assays have all been described.     

Platelet function testing by flow cytometry.

The availability of fluorescent monoclonal antibodies and probes now provides a powerful tool for examining platelet function by flow cytometry. These techniques cna be employed to determine the resting and stimulated expression of platelet glycoprotein receptors, the activation status of platelets assessed by secretion of granule contents (including expression of activation-dependent neoantigens), adhesion ligand binding to platelets, and intracellular calcium flux after exposure of platelets to agonist. In addition, flow cytometry has now been used to study the functional properties of stimulated platelets, including microparticle generation, platelet aggregation, and platelet-heterotypic cell conjugate formation. This brief review is presented as a general outline of the literature that uses flow cytometric methodology to examine in vivo and ex vivo platelet function.     

Dual parameter flow cytometry studies in human lymphomas.

Dual parameter flow cytometry studies using Coulter volume and cell DNA content were carried out in monodisperse cell suspensions of 64 samples of human lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, and benign lymphoid proliferations. Differences in mean Coulter volume among the lymphomas were due both to the intrinsic differences in mean G1 cell Coulter volume and to the presence of increased fractions of larger S and G2 cells, especially among the large B cell lymphomas. However, the relative contribution of large non-G1 cells to the overall population Coulter volume distribution was a relatively minor one; the presence of cells in S did not increase mean Coulter volume by more than 10%, even in samples with high S fractions. There was a good correlation between mean G1 cell Coulter volume and the log of the fraction of cells in S among the B cell lymphomas (r = 0.55). Evidence is presented that within individual samples, large cells proliferate more rapidly than small cells. This was seen in every case, both in the normal samples and in the lymphomas, and in the T cell lymphomas as well as in the B cell lymphomas. Aneuploidy was detected by flow cytometry in 11 cases; in 7 cases the aneuploid cell component could be analyzed separately from the diploid cell component on the basis of cell Coulter volume differences. The aneuploid components of diploid-aneuploid mixtures had higher S fractions than the diploid components in six of seven cases (0.16 +/- 0.04 [SE] vs. 0.08 +/- 0.02). These findings are considered in relation to the histopathological classification of the lymphomas, and in relation to the concept of clonal selection and clonal evolution of tumors.     

Rare-event analysis in flow cytometry

Technical aspects of rare-event detection are discussed in this article in a practical context, with two real-life examples. A growing number of flow cytometry-based assays depend on rare-event detection for basic science and clinical applications.     

Utility of sonographically guided biopsy in metabolically suspected recurrent lymphoma.

OBJECTIVE: The purpose of this study was to determine the diagnostic accuracy of sonographically guided biopsy of [(18)F]fluorodeoxyglucose (FDG)-avid foci on positron emission tomography (PET)/computed tomography (CT) in patients with lymphoma. METHODS: We retrospectively reviewed the medical records of 56 patients with lymphoma (25 male and 31 female; mean age, 48.5 years; range, 22-80 years) who underwent sonographically guided biopsy of hypermetabolic FDG-avid foci precisely localized by PET/CT. Biopsies were performed up to 3 months after PET/CT. The accuracy of core biopsy was calculated and compared with clinical follow-up and histopathologic results of open biopsy. RESULTS: Sixty-six sonographically guided biopsies were performed in the 56 patients. Histopathologic results were conclusive in 53 (80%) of 66. No complications occurred during or after the procedure. The overall sensitivity, specificity, positive predictive value, and accuracy for diagnosis of lymphoma were 100%, 95%, 97%, and 98%, respectively. CONCLUSIONS: Sonographically guided biopsy is a safe and effective means for investigating metabolically active lesions on FDG-PET/CT in patients with known lymphoma.