Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry.

Human immunodeficiency virus type-1 (HIV-1) DNA and messenger RNA sequences in both cell lines and blood obtained directly from HIV-1-infected patients were amplified by polymerase chain reaction and hybridized to fluorescein-labeled probes in situ, and the individually labeled cells were analyzed by flow cytometry. After flow cytometric analysis, heterogeneous cell populations were reproducibly resolved into HIV-1-positive and -negative distributions. Fluorescence microscopy showed that the cellular morphology was preserved and intracellular localization of amplified product DNA was maintained. Retention of nonspecific probe was not observed. Analysis of proviral DNA and viral messenger RNA in cells in the blood of HIV-1-infected patients showed that the HIV-1 genome persists in a large reservoir of latently infected cells. With the use of this technique it is now possible to detect single-copy DNA or low-abundance messenger RNA rapidly and reproducibly in a minor subpopulation of cells in suspension at single-cell resolution and to sort those cells for further characterization.

Phase I/II study of combined granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor administration for the mobilization of hematopoietic progenitor cells.

PURPOSE: To study the toxicity and efficacy of combined granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) administration for mobilization of hematopoietic progenitor cells (HPCs). MATERIALS AND METHODS: Cohorts of a minimum of five patients each were treated subcutaneously as follows: G-CSF 5 micrograms/kg on days 1 to 12 and GM-CSF at .5, 1, or 5 micrograms/kg on days 7 to 12 (cohorts 1, 2, and 3); GM-CSF 5 micrograms/kg on days 1 to 12 and G-CSF 5 micrograms/kg on days 7 to 12 (cohort 4); and G-CSF and GM-CSF 5 micrograms/kg each on days 1 to 12 (cohort 5). Ten-liter aphereses were performed on days 1 (baseline, pre-CSF), 5, 7, 11, and 13. Colony assays for granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) were performed on each harvest. RESULTS: The principal toxicities were myalgias, bone pain, fever, nausea, and mild thrombocytopenia, but none was dose-limiting. Four days of treatment with either G-CSF or GM-CSF resulted in dramatic and sustained increases in the numbers of CFU-GM per kilogram collected per harvest that represented 35.6 +/- 8.9- and 33.7 +/- 13.0-fold increases over baseline, respectively. This increment was attributable both to increased numbers of mononuclear cells collected per 10-L apheresis and to increased concentrations of progenitors within each collection. The administration of G-CSF to patients already receiving GM-CSF (cohort 4) caused the HPC content to surge to nearly 80-fold the baseline (P = .024); the reverse sequence, ie, the addition of GM-CSF to G-CSF, was less effective. The CFU-GM content of the baseline aphereses correlated with the maximal mobilization achieved (r = .74, P = .001). CONCLUSION: Combined G-CSF and GM-CSF administration effectively and predictably mobilizes HPCs and facilitates apheresis.

Flow cytometric DNA analysis of carcinoid tumors of the ileum and appendix.

Although all carcinoid tumors are considered potentially malignant, the biologic behaviors of appendiceal and ileal carcinoids are distinctly different. Appendiceal carcinoids often behave in a benign fashion, whereas ileal carcinoids pursue an aggressive course with frequent metastasis. Whether differences in DNA ploidy are related to this disparity in tumor behavior was addressed in this study. Flow cytometric DNA analyses were performed on paraffin blocks from 11 ileal and seven appendiceal carcinoid tumor cases. The mean coefficient of variation for all samples was 3.4 +/- 0.7. DNA aneuploidy was seen in two of the appendiceal cases and in six of the ileal cases. Metastases were seen in one of the appendiceal carcinoid cases, and that tumor was aneuploid. In six cases of carcinoid of the ileum, metastases were seen; of these, five tumors were aneuploid. In the ileal cases, despite the low number of cases examined, the correlation between DNA aneuploidy and metastases nearly reached statistical significance (P = .07) and showed a much stronger correlation than tumor size and metastases (P = .4). Although no statistical significance was reached in this study, the results are highly suggestive of DNA aneuploidy being an important predictor of malignant behavior in carcinoids of the ileum.

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.

DNA flow cytometric analysis of hemangiopericytoma.

The biologic behavior of hemangiopericytoma is difficult to predict using clinical or histologic criteria. The authors studied 22 hemangiopericytomas (including "angioblastic meningiomas") from 16 patients. Included in the study were six recurrent tumors and one metastatic tumor. DNA flow cytometric analysis was performed on 21 tumors for which paraffin-embedded material was available. All of the tumors were DNA diploid. However, among patients with adequate follow-up information, all tumors that exhibited aggressive behavior (local recurrence, metastasis, death due to invasive disease) had S-phase fractions of greater than 9% and proliferative indices (S-phase plus G2M phase) of greater than 11%. There was also a trend toward aggressive behavior in tumors with necrotic foci. Tumors arising in the central nervous system behaved more aggressively than primary tumors at other sites. This study showed a trend toward more aggressive behavior in hemangiopericytomas with higher proliferative indices. DNA ploidy, however, was not a useful indicator of biologic behavior in these tumors.

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.

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.

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.

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.

Karyotype evolution in a simian virus 40-transformed tumorigenic human cell line.

Normal human foreskin fibroblasts (HSF4) were transfected using the pSV3-neo plasmid. A pool of 10 G418-resistant colonies, HSF4-T12, showed a progressive increase in the expression of a number of in vitro transformation markers with passage in culture and became immortalized. Although no tumors were formed when cells were injected subcutaneously into nude mice, this cell line produced progressive tumors when cells were injected into preimplanted Gelfoam sponges in the mice. When these tumors were cultured in vitro and subsequently injected subcutaneously, progressive tumors were produced with median latency periods as short as 4 weeks. Three phases of cytogenetic change could be distinguished. At early passages after transfection. HSF4-T12 exhibited many random chromosomal changes. At a time just after immortalization, both flow karyotype and G-banded analyses showed the appearance of balanced clonal rearrangements. These included t(2;4), t(2;14), t(3;?), 6p-, i(6p), 8p-, t(14;15), i(15), and t(18;?). These clonal rearrangements were stable with passage in culture, and less variability from cell to cell was noted. The only consistent chromosomal loss observed was -Y. Analysis of three independent tumors showed characteristic loss of chromosomal material rather than balanced chromosomal rearrangements. Frequent loss of 6q and chromosomes #13, 15, 20, and Y was noted.

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.

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.

Leukocyte adhesion molecules as biocompatibility markers for hemodialysis membranes.

Comparative flow cytometric measurement was used to evaluate the significance of leukocyte adhesion molecule (LAM) activity changes during hemodialysis (HD) with different cellulosic and non cellulosic membranes. Six hemodialysis patients (men) who were in a maintenance program for more than 6 months were treated consecutively with five different dialyzers (cuprophan, hemophan, 2 types of cellulose acetate, and polysulfone). During each study HD, blood was sampled from the arterial line at 0, 15, and 60 min and from the venous port at 3 min to harvest leukocytes immediately after the first cell-membrane contact. After whole blood lysis preparation, leukocytes were incubated with fluorescent antibodies to label LAM CD 11A/18 (LFA-1), CD 11B/18 (Mac-1), CD 11C/18 (p150/95), and CD 54 (ICAM-1) (Becton-Dickinson, San Jose, CA). Data were acquired for the granulocyte, monocyte, and lymphocyte population based on forward and 90 degrees scatter light measurements. Accuracy of gating was verified by CD 14/45 staining for all samples. Baseline integrin expression for the selected populations before biomaterial contact was found to be heterogeneous for different patients, but underwent changes for the same patient during HD treatment. The fluorescent intensity corresponding to specific integrins was characterized by different patterns of up/down regulation with maximal deviations occurring at 3 min. Fluorescent intensity of the granulocyte and monocyte populations sampled at 15 min was 40-50% lower as compared with those sampled immediately after the first biomaterial contact. Based on the basal fluorescence levels and values recorded after the first biomaterial contact and those at 15 min, two coefficients were generated to compare membrane properties.

Multivariate analyses of cognitive and cognitive style variables based on hemisphere specialization theory predictive of success in a college developmental studies program.

Right and left cerebral hemisphere and limbic scores derived from the Herrmann Brain Dominance Profile, Scholastic Aptitude Test Verbal and Mathematics scores, and High School Grade Point Average were correlated with grades in college developmental courses in reading, English, and mathematics for 146 students. Pearson correlations ranged from -.27 to .42. Multiple correlations with seven predictors ranged from .45 to .55, and from .14 to .37 for the profile scores alone. Discriminant analyses yielded hit-rates (predictive of classification of success and failure/actual classification) of 69%/72% in Reading, 76%/75% in English, and 66%/71% in Mathematics.

Cell bioprocessing in space: applications of analytical cytology.

Cell bioprocessing in space consists of the preparation, cultivation, purification and investigation of cells and their products in the microgravity environment of orbital space flight. Inertial acceleration is used as an independent variable to explore the limits of specific bioprocessing functions, such as cell growth and secretion, gravity-dependent phenomena in cell bioreactors, cell fusion, the influence of thermal convection on processes at cellular dimensions, the electrophoretic separation of cell subpopulations and subcellular particles, and two-phase partitioning of cells, bioparticles, and macromolecules. Analytical cytology techniques are under development for on-orbit application to future cell growth and separation experiments, such as those anticipated in the Space Station era.

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.

TIMP-2, a growth-stimulatory protein from SV40-transformed human fibroblasts.

The mechanisms by which the early genes of simian virus 40 (SV40) transform human cells are unclear; however, this is clearly a multistep process involving a number of cellular and genetic changes. An early change following expression of the SV40 genes is growth under reduced serum conditions, which could be consistent with the production of autocrine/paracrine growth factors. HSF4-T12 is a human fibroblast cell line produced by transfection of primary cells with the genes for large T and small t antigens. A progressive stepwise transformation was observed with in vitro culture, eventually resulting in a tumorigenic cell line. Serum-free defined medium conditioned by HSF4-T12 was able to stimulate growth of normal human fibroblasts as determined by growth curve and [3H]-thymidine incorporation assays. Purification of this activity by heparin affinity chromatography and nondenaturing polyacrylamide gel electrophoresis resulted in a single band of approximately 21 kDa on a nonreducing, denaturing gel. A partial 14-amino acid sequence was found to share 100% homology with a region of tissue inhibitor of metalloproteinases-2 (TIMP-2). Western blot analysis with anti-TIMP-2 antiserum confirmed this identification, and addition of this same antiserum to HSF4-T12-conditioned medium resulted in inhibition of stimulatory activity.