Flow Cytometry Staff

The Flow Cytometry Unit provides specialized diagnostic procedures utilizing cytometric techniques as a clinical service. The laboratory immunophenotypes patient specimens from adult and pediatric Clinical Center patients with lymphoid neoplasms, acute as well as chronic leukemias, myelodysplastic syndrome, myeloproliferative syndrome, acquired immunodeficiency syndrome, and congenital immunodeficiency disorders. The majority of specimens receive their primary surgical pathology diagnosis within the unit (morphological evaluation is also performed by the unit). These specimens provide diagnostic challenges and an exceptional opportunity for clinical research in the field of flow cytometry.

Flow Cytometric immunophenotyping is a sensitive technique for analysis of benign and malignant tumors. We are studying the refinement of this technique and its application to diagnosis and measurement of prognostic markers in different systems. The myelodysplastic syndromes (MDS) are characterized by bi- or trilineage dysplasia. Although diagnostic criteria are well established for MDS, a significant number of patients have blood and bone marrow findings that make diagnosis and classification difficult. Flow cytometric immunophenotyping is an accurate and highly sensitive method for detection of quantitative and qualitative abnormalities in hematopoietic cells. We used flow cytometry to study hematopoietic cell populations in the bone marrow of patients with straightforward MDS. The results were compared to those obtained in a series of patients with aplastic anemia, normal donors and patients with a history of non-myeloid neoplasia in complete remission. We defined the immunophenotypic abnormalities associated with MDS and compared the diagnostic utility of flow cytometry with morphological and cytogenetic evaluations in difficult cases. Although morphology and cytogenetics were adequate for diagnosis in the majority of cases, flow cytometry could detect immunophenotypic abnormalities in cases when combined morphology and cytogenetics were non-diagnostic. We conclude that flow cytometric immunophenotyping may help establish the diagnosis of MDS, especially when morphology and cytogenetics are indeterminate.

The Flow Cytometry Unit conducts translational research into mechanisms of lymphomagenesis. Homeostasis in lymphoid tissues is maintained by close regulation of lymphoid cell proliferation and programmed cell death, or apoptosis. Inhibition of apoptosis can result in a neoplastic expansion of lymphoid cells (i.e., lymphoma) due to unchecked proliferation. In addition, genetic events leading to inhibition of programmed cell death result in resistance to numerous chemotherapeutic regimens as well as gamma-radiation. Factors that inhibit apoptosis can therefore result in formation of lymphomas with poor prognosis due to resistance to treatment.

The tissue inhibitors of metalloproteinases (TIMP's) are a family of closely related proteins that were initially described as inhibitors of the matrix metalloproteinases. We have shown that in addition to blocking MMP activity, TIMPs also have MMP independent growth factor-like activity in B-cells. TIMP-1 is expressed by reactive lymphoid cells as well as cell lines derived from B-cell neoplasms. TIMP-1 expression in these cells is unrelated to MMP expression. TIMP-1 expression correlates with differentiation state and appears to be expressed by a specific stage of germinal center B-cells. TIMP-1 induces further differentiation in B-cells to the germinal center phenotype that occurs with the generation of lymphoblasts. TIMP-1 expression also correlates positively with Group II/III EBV latency phenotype. TIMP-1 affects B-cell activation by inducing expression and secretion of the activation marker CD23. TIMP-1 inhibits cold shock, Fas, radiation and serum starvation induced apoptosis without causing withdrawal from cell cycle. TIMP-1 up-regulates the survival antigen CD40 and down-regulates expression of CD77, a neutral glycolipid expressed by a subset of B lymphocytes that readily enter programmed cell death. TIMP-1 up-regulates Bcl-XL but does not affect Bcl-2 or Mcl-1 expression. TIMP-1 also does not modify cytoplasmic levels of NF-kB but does increase expression of the NF-kB inhibitor IkBa. We have shown that EBV induces expression of TIMP-1 in B-cells and are studying the meachnism. Interleukin-10 (IL-10) is expressed by non-Hodgkin's lymphomas, where it acts as a cooperative growth factor. TIMP-1 induces IL-10 expression in B-cells in a non-MMP dependent manner. IL-10 does not protect the cells from induction of apoptosis nor induce the further B-cell differentiation. These actions are specific to TIMP-1 and occur in the absence of active IL-10. Furthermore, IL-10 induces proliferation. Therefore, TIMP-1 directly inhibits apoptosis and indirectly stimulates proliferation by inducing expression of IL-10. In a study of B-cell non-Hodgkin's lymphomas there was a high degree of correlation between TIMP-1 expression, histologic grade and IL-10 expression.
In summary, TIMP-1 induces B-cell differentiation, proliferation and inhibits apoptosis. Furthermore, TIMP-1 expression may be a negative prognostic factor in non-Hodgkin's lymphoma.

Recent Publications:
Xie, X.Y., et al. Leukemia and Lymphoma 37:97-104, 2000
Xie, X.Y., et al, Cytometry , 42:114-117, 2000.
Guedez, L, et al. Blood. 97:1796-1802, 2001
Stetler-Stevenson, M., et al. Blood 98: 979-987, 2001
Kingma, D.W. et al, Case Studies in Clinical Flow Cytometry 1(1):2001.

Collaborators:
Jerrold M. Ward, D.V.M., Ph.D.; William G. Stetler-Stevenson, M.D., Ph.D.; and Lionel Feigenbaum, Ph.D. NCI, NIH
Gerald E. Marti, M.D., Ph.D., Food and Drug Administration
Robert Hawley, Ph.D., Red Cross, Rockville, MD.