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Flow cytometric detection of the classical hodgkin lymphoma: clinical and research applications.

Roshal M, Wood BL, Fromm JR - Adv Hematol (2010)

Bottom Line: Recently, a reliable flow cytometric assay for direct detection and isolation of the malignant cells in this disease has been developed.This assay has proven useful diagnostically and has been clinically validated to have a very high sensitivity and nearly absolute specificity for the diagnosis of CHL in routine clinical samples.The current state of flow cytometric evaluation of nodular lymphocyte predominant Hodgkin lymphoma and T cell-rich large B cell lymphoma is also briefly discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
Classical Hodgkin lymphoma (CHL) is a relatively uncommon B cell-derived neoplasm that presents with rare malignant cells in an abundant reactive background. The diagnosis of CHL currently relies on a combination of morphologic findings and immunohistochemical stains. With the exception of rare cases with dramatically increased malignant populations, isolation of pure viable tumor cells has not been historically possible. Recently, a reliable flow cytometric assay for direct detection and isolation of the malignant cells in this disease has been developed. This assay has proven useful diagnostically and has been clinically validated to have a very high sensitivity and nearly absolute specificity for the diagnosis of CHL in routine clinical samples. This paper describes the methodology for the flow cytometric detection of CHL in clinical samples as well as current state of evaluation of background lymphocytes as an adjunct diagnostic test. Also discussed are exciting research applications of the direct isolation of viable tumor cells in CHL. The current state of flow cytometric evaluation of nodular lymphocyte predominant Hodgkin lymphoma and T cell-rich large B cell lymphoma is also briefly discussed.

No MeSH data available.


Related in: MedlinePlus

Identification of a small HRS population in a clinical sample:  malignant HRS cells shown in red have increased forward scatter area (FSC-A) and height (FSC-H) (note a subpopulation with HRS cells with disproportionately increased FSC-A likely corresponding to a rosetted population) and increased side scatter height (SSC-H) compared to the rest of the node cellularity (blue). The population showed bimodal, but mostly bright, expression of CD45, low levels of a B cell-marker CD20, no significant expression of a monocyte-marker CD64  (mild increase in apparent CD64 expression is due to autofluorescence of the HRS cells, which was previously verified by isotype control, data not shown), bright CD30, mostly bright CD5 (with a noticeable nonrosetted CD5 low to negative population), bright CD15 at a level slightly lower than granulocytes, and bright CD71 (transferrin receptor) consistent with high metabolic activity. Finally, the population shows a tight cluster in multiple projections including CD95 (bright) versus  CD40. In this case, the HRS population represented 0.09% of the total white cells in the lymph node. The case was morphologically confirmed as CHL-nodular sclerosis type.
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fig3: Identification of a small HRS population in a clinical sample: malignant HRS cells shown in red have increased forward scatter area (FSC-A) and height (FSC-H) (note a subpopulation with HRS cells with disproportionately increased FSC-A likely corresponding to a rosetted population) and increased side scatter height (SSC-H) compared to the rest of the node cellularity (blue). The population showed bimodal, but mostly bright, expression of CD45, low levels of a B cell-marker CD20, no significant expression of a monocyte-marker CD64 (mild increase in apparent CD64 expression is due to autofluorescence of the HRS cells, which was previously verified by isotype control, data not shown), bright CD30, mostly bright CD5 (with a noticeable nonrosetted CD5 low to negative population), bright CD15 at a level slightly lower than granulocytes, and bright CD71 (transferrin receptor) consistent with high metabolic activity. Finally, the population shows a tight cluster in multiple projections including CD95 (bright) versus CD40. In this case, the HRS population represented 0.09% of the total white cells in the lymph node. The case was morphologically confirmed as CHL-nodular sclerosis type.

Mentions: Further development of a reliable method of diagnosis of classical Hodgkin lymphoma by flow cytometry has benefited from the advances in the instrumentation. Introduction of rapid digital event acquisition on the modern cytometers now allows for the routine analysis of 500,000 events or more within less than 5 minutes, bringing a population that represents 0.01% of the total white cells well within range of sensitivity of clinical cytometry. In addition, increased numbers of lasers and detectors on the modern instruments allow for simultaneous interrogation of 10 or more antigens within the same tube. This, in turn, increases confidence in identification of small populations based on multidimensional analysis of multiple antibody staining patterns on single cells [12]. In our practice, using a nine-antibody combination in a single tube (currently CD5-ECD, CD15-APC, CD20-PE-Cy7, CD30-PE, CD40-PE-Cy5.5, CD45-APC-H7, CD64-FITC, CD71-APC-A700, and CD95-PB) allows for detection of cases with a relatively low abundance of HRS cells even with some deviation from the canonical CD30 and CD15 bright immunophenotype [9, 10]. Such detection relies on stepwise exclusion of reactive populations (CD20-positive B cells, CD5-positive T cells, and CD64-positive monocytes and granulocytes) and refinement of the population of interest with the most common immunophenotype of CD15 (intermediate to bright), CD30 (intermediate to bright), CD40 (bright), CD71 (bright), and CD95 (bright) in multiple projections. Figure 3 demonstrates the usage of this strategy to detect a relatively small malignant population in a CHL lymph node.


Flow cytometric detection of the classical hodgkin lymphoma: clinical and research applications.

Roshal M, Wood BL, Fromm JR - Adv Hematol (2010)

Identification of a small HRS population in a clinical sample:  malignant HRS cells shown in red have increased forward scatter area (FSC-A) and height (FSC-H) (note a subpopulation with HRS cells with disproportionately increased FSC-A likely corresponding to a rosetted population) and increased side scatter height (SSC-H) compared to the rest of the node cellularity (blue). The population showed bimodal, but mostly bright, expression of CD45, low levels of a B cell-marker CD20, no significant expression of a monocyte-marker CD64  (mild increase in apparent CD64 expression is due to autofluorescence of the HRS cells, which was previously verified by isotype control, data not shown), bright CD30, mostly bright CD5 (with a noticeable nonrosetted CD5 low to negative population), bright CD15 at a level slightly lower than granulocytes, and bright CD71 (transferrin receptor) consistent with high metabolic activity. Finally, the population shows a tight cluster in multiple projections including CD95 (bright) versus  CD40. In this case, the HRS population represented 0.09% of the total white cells in the lymph node. The case was morphologically confirmed as CHL-nodular sclerosis type.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Identification of a small HRS population in a clinical sample: malignant HRS cells shown in red have increased forward scatter area (FSC-A) and height (FSC-H) (note a subpopulation with HRS cells with disproportionately increased FSC-A likely corresponding to a rosetted population) and increased side scatter height (SSC-H) compared to the rest of the node cellularity (blue). The population showed bimodal, but mostly bright, expression of CD45, low levels of a B cell-marker CD20, no significant expression of a monocyte-marker CD64 (mild increase in apparent CD64 expression is due to autofluorescence of the HRS cells, which was previously verified by isotype control, data not shown), bright CD30, mostly bright CD5 (with a noticeable nonrosetted CD5 low to negative population), bright CD15 at a level slightly lower than granulocytes, and bright CD71 (transferrin receptor) consistent with high metabolic activity. Finally, the population shows a tight cluster in multiple projections including CD95 (bright) versus CD40. In this case, the HRS population represented 0.09% of the total white cells in the lymph node. The case was morphologically confirmed as CHL-nodular sclerosis type.
Mentions: Further development of a reliable method of diagnosis of classical Hodgkin lymphoma by flow cytometry has benefited from the advances in the instrumentation. Introduction of rapid digital event acquisition on the modern cytometers now allows for the routine analysis of 500,000 events or more within less than 5 minutes, bringing a population that represents 0.01% of the total white cells well within range of sensitivity of clinical cytometry. In addition, increased numbers of lasers and detectors on the modern instruments allow for simultaneous interrogation of 10 or more antigens within the same tube. This, in turn, increases confidence in identification of small populations based on multidimensional analysis of multiple antibody staining patterns on single cells [12]. In our practice, using a nine-antibody combination in a single tube (currently CD5-ECD, CD15-APC, CD20-PE-Cy7, CD30-PE, CD40-PE-Cy5.5, CD45-APC-H7, CD64-FITC, CD71-APC-A700, and CD95-PB) allows for detection of cases with a relatively low abundance of HRS cells even with some deviation from the canonical CD30 and CD15 bright immunophenotype [9, 10]. Such detection relies on stepwise exclusion of reactive populations (CD20-positive B cells, CD5-positive T cells, and CD64-positive monocytes and granulocytes) and refinement of the population of interest with the most common immunophenotype of CD15 (intermediate to bright), CD30 (intermediate to bright), CD40 (bright), CD71 (bright), and CD95 (bright) in multiple projections. Figure 3 demonstrates the usage of this strategy to detect a relatively small malignant population in a CHL lymph node.

Bottom Line: Recently, a reliable flow cytometric assay for direct detection and isolation of the malignant cells in this disease has been developed.This assay has proven useful diagnostically and has been clinically validated to have a very high sensitivity and nearly absolute specificity for the diagnosis of CHL in routine clinical samples.The current state of flow cytometric evaluation of nodular lymphocyte predominant Hodgkin lymphoma and T cell-rich large B cell lymphoma is also briefly discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
Classical Hodgkin lymphoma (CHL) is a relatively uncommon B cell-derived neoplasm that presents with rare malignant cells in an abundant reactive background. The diagnosis of CHL currently relies on a combination of morphologic findings and immunohistochemical stains. With the exception of rare cases with dramatically increased malignant populations, isolation of pure viable tumor cells has not been historically possible. Recently, a reliable flow cytometric assay for direct detection and isolation of the malignant cells in this disease has been developed. This assay has proven useful diagnostically and has been clinically validated to have a very high sensitivity and nearly absolute specificity for the diagnosis of CHL in routine clinical samples. This paper describes the methodology for the flow cytometric detection of CHL in clinical samples as well as current state of evaluation of background lymphocytes as an adjunct diagnostic test. Also discussed are exciting research applications of the direct isolation of viable tumor cells in CHL. The current state of flow cytometric evaluation of nodular lymphocyte predominant Hodgkin lymphoma and T cell-rich large B cell lymphoma is also briefly discussed.

No MeSH data available.


Related in: MedlinePlus