In situ hybridization in flow cytometry.

Molecular approaches to diagnostic questions in clinical medicine are greatly impacting the way researchers and clinicians investigate and treat disease. By combining molecular techniques with classical immunologic tools such as flow cytometry (FCM; 1-3), one can begin to more fully understand and appreciate the role of cellular heterogeneity in disease processes. The marriage of these two powerful techniques, termed molecular cytometry, will, in one instance, allow investigators to explore expression of nucleic acid sequences in subpopulations of cells defined by immunologic phenotype while, conversely, making it possible to examine the heterogeneity of cellular characteristics within populations identified by the presence of specific nucleic acid sequences or gene expression. Future developments may result in several advantages for the patient that may include, but are not limited to, earlier detection of viral infection, earlier and more sensitive detection of malignancy, and higher sensitivity and resolution of small populations of infected or aberrant cells. These developments may also assist in the identification of therapeutically resistant populations within a neoplasia, more effective and specific monitoring of therapy, and possibly the identification of new and disease-specific targeted therapies based on genetic information. The characterization and assessment of cellular heterogeneity is clearly key to understanding disease onset, progression, and therapeutic response in both infectious disease and in human malignancies.

Lymphocyte subsets and activation markers in patients with acute episodes of idiopathic anaphylaxis.

BACKGROUND: Idiopathic anaphylaxis (IA), a type of anaphylaxis in which no external allergen can be identified, is a corticosteroid-responsive disease, that suggests that it may have an immunologic pathogenesis. OBJECTIVE: The objective of this study is to compare patients with acute episodes of IA with normals, patients with chronic idiopathic urticaria, and patients with IA in remission relative to lymphocyte subsets and activation markers. METHODS: This is a prospective cohort study of 38 adults: 5 normals, 4 idiopathic urticaria, 11 IA patients in remission, 9 IA patients with acute attacks who had not yet received prednisone, and 9 IA patients who had received prednisone. The main outcome measures were lymphocyte subset and activation markers determined by two and three color flow cytometry (CD2, CD3, CD4, CD5, CD8, CD16, CD19, CD23, CD25, CD56, and HLA-DR). RESULTS: Comparing patients with acute IA with those in remission, the only significant difference was that the acute IA patients had a significantly higher percentage of CD3+HLA-DR+ cells. Normals had a significantly lower percentage of CD3+ HLA-DR+ cells than all other groups. Patients with acute IA on prednisone as well as IA patients in remission had a significantly higher percentage of CD 19+ CD23+ cells than normals. CONCLUSIONS: These results suggest that there are more activated T cells in patients with acute episodes of IA than in patients in remission. Perhaps, these activated T cells have a role in the pathogenesis of IA.

Atypical hairy cell leukemia.

The morphologic differential diagnosis of mature B-cell neoplasms with cytoplasmic projections includes splenic lymphoma with villous lymphocytes and hairy cell leukemia. Although the classification of hairy cell leukemia is not universally recognized, 3 variants have been described, namely, classic, variant, and Japanese variant, each of which has different clinical and immunophenotypic features. Classic hairy cell leukemia is virtually always CD11c(+), CD25(+), and CD103(+). Variant and Japanese variant hairy cell leukemias are usually CD11c(+), always CD25(-), and occasionally CD103(+). Each variant is characteristically CD10(-). We present a case of hairy cell leukemia with a unique immunoprofile in that the cells were CD10(+), CD25(+), and CD103(-), and we review the criteria helpful in differentiating "hairy" B-cell neoplasms. This case emphasizes the variability of hairy cell leukemia and the need to correlate all clinical and pathologic data in reaching a diagnosis.

Estrogen and progesterone receptors in gynecomastia.

The etiology of gynecomastia is unknown. There seems to be no increased incidence of malignancies in patients with idiopathic gynecomastia; however, patients with Klinefelter syndrome exhibit an increased incidence of malignancy. The authors reviewed the results of 34 patients with gynecomastia diagnosed in adolescence who, following initial evaluation, had a mastectomy. The estrogen and progesterone receptors were analyzed in these patients. Three of the patients were diagnosed with Klinefelter syndrome. These three patients exhibited elevated amounts of estrogen and progesterone receptors. None of the patients who were not diagnosed with this syndrome demonstrated significant elevation of their estrogen or progesterone receptors. The presence of elevated estrogen and progesterone receptors in patients with Klinefelter syndrome provides a potential mechanism by which these patients may develop breast neoplasms. The absence of elevated estrogen and progesterone receptors in patients with idiopathic gynecomastia may serve to clarify why these patients' disease rarely degenerates into malignancy.

Expansions of clonal and oligoclonal T cells in B-cell chronic lymphocytic leukemia are primarily restricted to the CD3(+)CD8(+) T-cell population.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of mature-appearing clonal B cells exhibiting coexpression of CD5 and CD23. In addition to the accumulation of neoplastic B cells, numerous T-cell abnormalities also occur in B-CLL patients. In this study, the presence, and distribution within the T-cell subsets, of clonal/oligoclonal T cells was studied. Multicolor flow cytometric techniques were employed using combinations of anti-CD3, anti-CD4, and anti-CD8 antibodies coupled with antibodies specific for V(alpha) and V(beta) T-cell receptor (TCR) epitopes. Molecular studies of TCR gene sequences were done to confirm the presence of clonal/oligoclonal T-cell populations. In the flow cytometric studies, examination of V(alpha)/V(beta)expression found evidence of clonal/oligoclonal expansion in 9 of 19 patients studied. In eight of the nine patients, the expansions were restricted to the CD3(+)CD8(+) cell population. Molecular analyses were performed in 16 patients, 12 of whom showed a clonal or oligoclonal pattern. Of the four patients who were negative in the molecular analyses, all demonstrated flow cytometric evidence of clonal/oligoclonal expansions. Thus, when the flow cytometric and molecular analyses were considered together, all 16 patients for whom parallel analyses were done showed evidence of clonal/oligoclonal expansions. These results confirm previous work demonstrating that the majority of B-CLL patients harbor clonal/oligoclonal expansions within the T-cell population. Additionally, based on the relative numbers of cells expressing specific V(alpha) or V(beta)epitopes, these results show that these expansions occur primarily within the CD3(+)CD8(+) T-cell population.

Lymphoid aggregates in bone marrow mimic residual lymphoma after rituximab therapy for non-Hodgkin lymphoma.

Rituximab is a novel anti-CD20 monoclonal antibody used in the treatment of relapsed low-grade non-Hodgkin lymphoma. To determine the impact of this therapy on the interpretation of posttherapy specimens, we reviewed the pretherapy and posttherapy bone marrow and peripheral blood morphologic and flow cytometric findings for 20 patients who received rituximab. Nine patients had a total of 13 posttherapy bone marrow specimens; all were positive for lymphoma before therapy. After therapy, 11 of 13 posttherapy bone marrow specimens were interpreted as positive or suggestive of lymphoma based on routine H&E-stained sections. However, immunohistochemical and/or flow cytometric immunophenotyping showed that 6 of the 11 cases were negative for lymphoma; the lymphoid infiltrates were composed entirely of T cells without B cells. We report that posttherapy bone marrow specimens from patients treated with rituximab may mimic residual lymphoma if examined by morphologic features alone. Familiarity with this finding and the use of ancillary immunophenotypic studies will aid in the accurate interpretation of posttherapy specimens.

CD10 antigen expression correlates with the t(14;18)(q32;q21) major breakpoint region in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a common morphologic term for a biologically diverse group of lymphomas. The chromosome translocation, t(14;18)(q32;q21), and its associated bcl-2 gene rearrangement are generally associated with follicular lymphomas. Some investigators, however, proposed that the presence of the t(14;18) in DLBCL suggests a possible follicle center cell origin and might correlate with a higher relapse rate after therapy. The CD10 antigen is expressed in a majority of follicular lymphomas but is also seen occasionally in DLBCLs. In this study, we examined 26 DLBCLs for CD10 expression by flow cytometric analysis and tested them for the t(14;18)(q32;q21) major breakpoint region by a polymerase chain reaction-based method. bcl-2 protein expression was analyzed by an immunoperoxidase method. Of the 26 DLBCLs, 9 (35%) were CD10 positive. bcl-2 protein was expressed in 7 (78%) of 9 CD10-positive cases and in 9 (53%) of 17 CD10-negative cases (P = .4). The t(14; 18) translocation was present in 6 (67%) of 9 CD10-positive cases but in only 2 (17%) of 12 CD10-negative cases (P = .03). Five cases did not yield amplifiable DNA for analysis. In summary, no difference in bcl-2 protein expression was seen in CD10-positive versus CD10-negative DLBCLs, but CD10-positive DLBCLs were significantly more likely than CD10-negative DLBCLs to harbor the t(14;18) translocation. This suggests that CD10 might be a marker of follicle center cell origin in DLBCL.

CD5- small B-cell leukemias are rarely classifiable as chronic lymphocytic leukemia.

Expression of the CD5 antigen by neoplastic cells often is considered a diagnostic criterion for B-cell chronic lymphocytic leukemia (B-CLL). However, published series frequently include a number of CD5- cases. We studied the spectrum of CD5- B-cell lymphoproliferative disorders presenting with leukemia involvement and reassessed the prevalence of CD5- B-CLL. We immunophenotyped 192 cases of clonal, small lymphocytic, B-cell disorders involving peripheral blood or bone marrow. Of these, 41 CD5- cases were further analyzed, correlating the immunophenotypic findings with pathologic material and clinical data. Only 3 CD5- cases were classified as CD5- B-CLL. These 3 cases had features unusual for B-CLL, including bright surface immunoglobulin expression, bright CD20 expression, and absence of CD23 expression (2 cases) or Richter syndrome (1 case). The remainder of the CD5- cases consisted of hairy cell leukemia, hairy cell variant, prolymphocytic leukemia, follicular center cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma (SMZL), small lymphocytic lymphoma with marrow fibrosis, and lymphoma, not further classified. Eight cases remained unclassified, but some displayed features of SMZL. CD5- lymphoproliferative disorders of peripheral blood or bone marrow are unlikely to be CLL and often are classified more appropriately as non-Hodgkin lymphoma in the leukemia phase.

Ex-vivo expansion of CFU-GM and BFU-E in unselected PBMC cultures with Flt3L is enhanced by autologous plasma.

BACKGROUND: Previous ex-vivo expansion studies in our laboratory, comparing unselected and CD34(+)-selected PBMC, have shown no advantage for CD34(+) cell selection, in terms of the expansion achieved. Our goal was to develop procedures for consistent generation of large numbers of hematopoietic progenitor and post-progenitor cells from unselected PBMC. METHODS: Unselected PBMC, collected from cancer patients undergoing apheresis prior to high-dose chemotherapy and autologous stem cell rescue, were expanded ex vivo in static cultures, without a stromal layer, in the presence of Flt3 ligand (Flt3L), a recombinant GM-CSF/IL-3 fusion protein (PIXY321), G-CSF and GM-CSF for 10 days. RESULTS: The addition of 2% autologous plasma to this cytokine combination enhanced expansion of total cell numbers (3.2 fold versus 1.9 fold; p < 0.01), colony-forming units granulocyte-macrophage (CFU-GM) (22.0 fold versus 8.1 fold, p < 0.01) and burst-forming units erythroid (BFU-E) (17.6 fold versus 7.0 fold, 0.01 < p < 0.02). The optimal seeding density for a given specimen was inversely related to the frequency of CD34(+) cells in the sample. CFU-GM expansion with the Flt3L-containing cytokine cocktail was equivalent to that obtained with IL-3, IL-6, G-CSF and SCF, whether or not the cultures were supplemented with autologous plasma. In plasma-free cultures, BFU-E expansion was significantly higher with IL-3, IL-6, G-CSF and SCF than with Flt3L, PIXY321, G-CSF and GM-CSF. In the presence of autologous plasma, however BFU-E expansion was higher in the Flt3L-containing media. In comparison studies, autologous plasma suppressed BFU-E expansion in SCF-containing cultures. Consistent with our colony assay results, dual-parameter flow cytometric analysis of the expanded cell population revealed that supplementation with autologous plasma yielded a significant increase in the numbers of myeloid progenitors in Flt3L-containing cultures. DISCUSSION: Unselected PBMC from cancer patients can be effectively expanded ex vivo in Flt3L, PIXY321, G-CSF and GM-CSF, supplemented with autologous plasma, yielding high numbers of myeloid and erythroid progenitors.

Treatment of autoimmune disease by intense immunosuppressive conditioning and autologous hematopoietic stem cell transplantation.

Multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis are immune-mediated diseases that are responsive to suppression or modulation of the immune system. For patients with severe disease, immunosuppression may be intensified to the point of myelosuppression or hematopoietic ablation. Hematopoiesis and immunity may then be rapidly reconstituted by reinfusion of CD34(+) progenitor cells. In 10 patients with these autoimmune diseases, autologous hematopoietic stem cells were collected from bone marrow or mobilized from peripheral blood with either granulocyte colony-stimulating factor (G-CSF) or cyclophosphamide and G-CSF. Stem cells were enriched ex vivo using CD34(+) selection and reinfused after either myelosuppressive conditioning with cyclophosphamide (200 mg/kg), methylprednisolone (4 g) and antithymocyte globulin (ATG; 90 mg/kg) or myeloablative conditioning with total body irradiation (1,200 cGy), methylprednisolone (4 g), and cyclophosphamide (120 mg/kg). Six patients with multiple sclerosis, 2 with systemic lupus erythematosus, and 2 with rheumatoid arthritis have undergone hematopoietic stem cell transplantation. Mean time to engraftment of an absolute neutrophil count greater than 500/microL (0.5 x 10(9)/L) and a nontransfused platelet count greater than 20,000/microL (20 x 10(9)/L) occurred on day 10 and 14, respectively. Regimen-related nonhematopoietic toxicity was minimal. All patients improved and/or had stabilization of disease with a follow-up of 5 to 17 months (median, 11 months). We conclude that intense immunosuppressive conditioning and autologous T-cell-depleted hematopoietic transplantation was safely used to treat these 10 patients with severe autoimmune disease. Although durability of response is as yet unknown, all patients have demonstrated stabilization or improvement.

Enhanced detection of malignant lymphoma in cerebrospinal fluid by multiparameter flow cytometry.

Immunophenotyping by flow cytometry has not been widely applied to cerebrospinal fluid (CSF) analysis. We attempted to optimize flow cytometric detection of malignant lymphoma in CSF samples by the routine use of 3- and 4-color flow cytometry, with specific selection of lymphoid cells by fluorescence vs 90 degrees light scatter gating. Thirty-six consecutive CSF samples were immunophenotyped by flow cytometry, and the results were compared with those of standard microscopic examination. Lymphoid events were adequate for analysis in 27 of the 36 samples. Each of the 9 unsuccessful samples was more than 24 hours old at analysis or contained fewer than 1 x 10(4) total cells (< or =1 cell/microL). Lymphoma was detected in 10 of the remaining 27 cases. Six lymphomas were detected by morphology and flow cytometry, 1 only by morphologic examination, and 3 only by flow cytometry. Therefore, the combination of flow cytometry and morphologic examination enhanced the detection by 43% over morphologic examination alone. Flow cytometry permitted the detection of lymphoid clones totaling less than 1% of total cells. Multicolor flow cytometry is a rapid and sensitive technique that enhances detection of lymphoma in paucicellular CSF samples. Given the great sensitivity of flow cytometry, future studies will be necessary to assess the significance of detecting small lymphoid clones in this setting.

T cell-depleted autologous hematopoietic stem cell transplantation for multiple sclerosis: report on the first three patients.

Multiple sclerosis (MS) is a disease of the central nervous system characterized by immune-mediated destruction of myelin. In patients with progressive deterioration, we have intensified immunosuppression to the point of myeloablation. Subsequently, a new hematopoietic and immune system is generated by infusion of CD34-positive hematopoietic stem cells (HSC). Three patients with clinical MS and a decline of their Kurtzke extended disability status scale (EDSS) by 1.5 points over the 12 months preceding enrollment and a Kurtzke EDSS of 8.0 at the time of enrollment were treated with hematopoietic stem cell (HSC) transplantation using a myeloablative conditioning regimen of cyclophosphamide (120 mg/kg), methylprednisolone (4 g) and total body irradiation (1200 cGy). Reconstitution of hematopoiesis was achieved with CD34-enriched stem cells. The average time of follow-up is 8 months (range 6-10 months). Despite withdrawal of all immunosuppressive medications, functional improvements have occurred in all three patients. We conclude that T cell-depleted hematopoietic stem cell transplantation can be performed safely in patients with severe and debilitating multiple sclerosis. Stem cell transplantation has resulted in modest neurologic improvements for the first time since onset of progressive disease although no significant changes in EDSS or NRS scales are evident at this time.

Detection of HIV-RNA-positive monocytes in peripheral blood of HIV-positive patients by simultaneous flow cytometric analysis of intracellular HIV RNA and cellular immunophenotype.

Determinations of plasma HIV viral RNA copy numbers help to define the kinetics of HIV-1 infection in vivo and to monitor antiretroviral therapy. However, questions remain regarding the identity of various infected cell types contributing to this free virus pool and to the in vivo lifecycle of HIV during disease progression. Characterization of a novel fluorescence in situ hybridization (FISH) assay employing a pool of labeled oligonucleotide probes directed against HIV RNA was done followed by coupling of the FISH assay with simultaneous surface immunophenotyping to address these questions. In vitro characterizations of this assay using tumor necrosis factor-alpha stimulated and unstimulated ACH-2 cells demonstrated the ability to detect < 5% HIV RNA positive cells with a sensitivity of < 30 RNA copies per cell. Peripheral blood mononuclear cells from 39 HIV-seropositive patients on no, single, combination, or triple drug therapy and 8 HIV-seronegative patients were examined. The majority of HIV-positive patients (24/39) harbored monocytes positive for HIV RNA and a significantly higher fraction of patients with high plasma viral load carried positive monocytes (13/16) than did patients in the low plasma viral load group (11/23). These results demonstrate the effectiveness of a novel FISH assay for identifying and monitoring HIV-infected cell populations in the peripheral blood of HIV-positive patients. In addition, monocytes are a major source of cellular HIV virus in the peripheral blood of HIV patients, even with progression of disease.

Reducing cellular autofluorescence in flow cytometry: an in situ method.

Cellular autofluorescence affects the sensitivity of flow cytometric assays by interfering with detection of low level specific fluorescence. These detection limits increase with use of protocols, such as thermocycling and fluorescent in-situ hybridization (FISH), that can increase intrinsic cellular fluorescence to 5,000-20,000 fluorescein isothiocyanate (FITC) equivalents. In order to improve signal to noise ratios when using FITC labeled probes in these procedures, we employed a method using the polyanionic azo dye, trypan blue, to reduce intracellular autofluorescence. Dyes such as these are commonly used in immunofluorescent microscopy to reduce background fluorescence. By using this method, we realized an approximately 5-fold increase in signal to noise ratio (S/N) in the direct detection of RNA target probes using flow cytometry. Trypan blue aided in the resolution of dim surface antibodies, internal markers and probes, and functions to reduce background autofluorescence after thermocycling and hybridization. This technique is rapid and easily applicable for reducing intracellular autofluorescence, and can be used in single and dual color applications.

In vitro production of megakaryocytes from PIXY321 versus GM-CSF-mobilized peripheral blood progenitor cells.

The generation of megakaryocytes (MK) from cultured peripheral blood progenitor cells (PBSC), harvested via apheresis, from 18 female breast cancer patients treated with either PIXY321 or GM-CSF was compared. Nonadherent mononuclear cells (MNC) were cultured in liquid suspension with 50 U/ml thrombopoietin (TPO) and 2.5% autologous heparinized plasma for 12 days. Flow cytometric analysis was used to measure the percentage of CD34+ on day 1 and CD41+ cells on day 12. The frequency of CD34+ cells was greater in GM-CSF-mobilized samples than in PIXY321-mobilized samples, and MK/MNC yields correlated directly with the number of CD34+ cells seeded, PIXY321-mobilized samples produced more MKs per CD34+ cell than GM-CSF-mobilized samples. Overall, there was no significant difference in the MK/MNC yield between PIXY321- and GM-CSF-mobilized samples. Cyclophosphamide (CY) increased the frequency of CD34+ cells and the corresponding MK/MNC yield for both cytokines, but had no effect on the MK/CD34+ yield. Compared to GM-CSF, PIXY321 mobilization resulted in increased CD34+ cell commitment to the MK lineage.

Karyotype correlates with peripheral blood morphology and immunophenotype in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is recognized as a distinct entity. However, morphologic and immunophenotypic heterogeneity exist. Twenty-six patients with CLL were studied to investigate whether an association exists among peripheral blood karyotype, morphology and immunophenotype. Clonal cytogenetic abnormalities were detected in 14 patients (53%), using conventional karyotyping techniques in addition to fluorescence in situ hybridization (FISH) for chromosome 12. By FAB guidelines, 7 of the 8 patients (88%) with trisomy 12 had mixed cell morphology compared to only 3 of 18 (17%) without trisomy 12 (P = .004). One patient (12%) with trisomy 12 had lymphocyte morphology typical for CLL. Six of the eight (75%) with trisomy 12 had atypical immunophenotype including one or more of the following: strong CD20 expression, strong surface light chain expression, or absence of CD23 expression. Only 2 of the 18 patients (11%) without trisomy 12 had atypical immunophenotype (P = .005). None of the three patients with clonal structural abnormalities of chromosome 13q14 had mixed cell morphology or atypical immunophenotype. One of the 12 patients (8%) without clonal cytogenetic abnormalities had mixed cell morphology and one had atypical immunophenotype. This study suggests that a correlation exists among karyotype, morphology, and immunophenotype in CLL, and that CLL subgroups can be identified based on laboratory parameters. Although normal karyotypes or clonal structural abnormalities of 13q14 are associated with morphology and immunophenotype considered typical for CLL, trisomy 12 is associated with mixed cell morphology and atypical immunophenotype. These findings may have implications for evaluating variation in both disease course and response to emerging therapies.

TIMP-2 growth-stimulatory activity: a concentration- and cell type-specific response in the presence of insulin.

In addition to proteinase-inhibitory activities, growth-stimulatory activities have been described for all three known members of the tissue inhibitors of the metalloproteinase (TIMP) family, TIMP-1, TIMP-2, and ChIMP-3, believed to be the chicken homologue of TIMP-3. However, the mechanism by which the TIMPs stimulate cell growth is unclear. In this report we have demonstrated that rTIMP-2 was growth-stimulatory for human foreskin fibroblasts (HSF4, HSF43, HS68), lung adenocarcinoma cells (A549), human melanoma cells (WM115), and the Burkitt's lymphoma cell line RAMOS, and this stimulatory response was concentration-dependent, with the greatest stimulation occurring a 10-30 pM rTIMP-2 in [3H]thymidine incorporation assays and at 20-100 pM in cell growth assays. Normal human colon (18Co) and lung (37Lu) fibroblasts showed no response to rTIMP-2. [3H]Thymidine incorporation was inhibited by rTIMP-2 treatment in the nonadherent cell line HL60. These studies also demonstrated that for the cell types tested, TIMP-2 alone was insufficient for a growth stimulatory response requiring, at a minimum, the presence of insulin. In the absence of any "co-factor(s)," such as insulin, TIMP-2 treatment was inhibitory.

Thrombopoietin-stimulated ex vivo expansion of human bone marrow megakaryocytes.

The effect of thrombopoietin (TPO) on megakaryocytopoiesis (MKP) has been mainly studied using clonogenic assays in murine systems. In this study, we evaluated MKP in liquid culture using human bone marrow cells. While interleukin 3 (IL-3) and stem cell factor (SCF) are potent activators of TPO-stimulated MKP in the murine system, only IL-3 exhibited synergistic activity with TPO in cultures of human bone marrow. The IL-3 effect on TPO-stimulated megakaryocyte (MK) proliferation, expressed as the absolute number of MKs per seeded CD34+ cell, was more pronounced with purified CD34+ cell (8 +/- 1.6 SE versus 2.8 +/- 0.7 SE in the presence and absence of IL-3, respectively) than with mononuclear cells (MNC) (16 +/- 2.8 SE versus 11 +/- 2.0 SE). This effect of IL-3 on TPO-stimulated MK proliferation was due to a general proliferation of all cell types since the relative frequency of MKs (32.1 +/- 3 SE and 55.8 +/- 3 SE in MNC and CD34+ cells, respectively) was not affected by IL-3. The effect of TPO alone, TPO + IL-3, TPO + SCF, and TPO + IL-3 + SCF on MK proliferation was examined in MNC and CD34+ cultures. Greater numbers of MK per seeded CD34+ were observed in MNC compared to CD34+ cultures under all conditions except when TPO was added with both IL-3 and SCF. The enhancing effect of MNC was also observed on MK ploidy in the presence of TPO and IL-3. While proliferation and ploidy increase with TPO concentration in the murine system, they are inversely related in the human system. A significant 2.5-fold enhancement of TPO-induced MK proliferation was observed when purified CD34+ cells were cultured in inserts separated from human bone marrow stroma, indicating that soluble stimulatory factors are released from the stroma. These observations will be useful for ex vivo expansion of MKs to treat post-transplant or chemotherapy-associated thrombocytopenia.

Flow cytometric analysis of neoplastic nodules and hepatocellular carcinomas induced by ciprofibrate in the rat.

Alterations in DNA ploidy accompany hepatocellular carcinoma (HCC). However, changes in DNA content are also seen in regenerating liver and with increasing age. Thus, to investigate the role of DNA ploidy changes in development of HCC, flow cytometric DNA content determinations were done in a rat model system of peroxisome proliferator-induced HCC. Paraffin blocks of liver isolated from 18 Fisher 344 male rats fed ciprofibrate for 20 weeks (4), 40 weeks (4) or 20 months (10) were examined. Livers from age-matched control rats were also examined. From the 20 month ciprofibrate group, nine neoplastic nodules (NNs), 27 HCCs and four non-tumorous surrounding tissue controls (NTCs) were examined. Significant DNA tetraploid populations were seen in both the NNs and NTCs. A significant increase in the percentage of DNA diploid cells was observed in the NN samples. No significant difference in the percentage S-phase cells was seen. Emergence of cell populations with new DNA ploidy classes (8c or DNA aneuploid) as compared with NTCs was only seen in HCCs (7 of 27), and five of these seven were DNA aneuploid, as distinct from DNA tetraploid, populations. A total of 16 of 24 HCC samples that were adequate for cell cycle analysis had average percent S-phase greater than the mean of the NTCs plus three standard deviations. Although a direct role cannot be inferred, these results support the hypothesis that increases in the fraction of diploid cells is an important early event in the development of rat HCC and that further alterations in DNA ploidy and increased proliferative fraction accompany the development of HCC.