Ifosamide, epirubicin and granulocyte colony-stimulating factor: a regimen for successful mobilization of peripheral blood progenitor cells in patients with multiple myeloma.

In general, the mobilization of peripheral blood progenitor cells (PBPC) in multiple myeloma (MM) patients is poor and is achieved in most cases by combined cyclophosphamide and G-CSF. This study was performed to examine the efficacy of combined ifosfamide/epirubicine and G-CSF for PBPC mobilization and purging. Sixteen patients suffering from multiple myeloma in stage II/A and III/A according to Durie and Salmon underwent chemotherapy consisting of a total of three cycles of ifosfamide (3 g/m(2) on days 1 and 2 and epirubicine 80 mg/m(2) on day 1) and G-CSF (10 or 20 microg/kg body weight (BW) daily until harvesting). PBPC harvesting was performed after the first and third cycle of chemotherapy. The median number of PBPC after the first cycle of chemotherapy was 7.79 x 10(6) CD34+ cells/kg BW (ranging from 0.94-26.36 x 10(6)) and 6.38 x 10(6) CD34+ cells/kg BW (ranging from 0.79-29.31 x 10(6)) after the third cycle of chemotherapy. Clinical re-evaluation after three cycles of chemotherapy showed 13 (81 per cent) patients in partial remission (PR), two (12 per cent) in complete remission (CR) and one (6.25 per cent) in stable disease (SD). No major side-effects were observed, six patients developed hematological toxicity stage IV WHO for a median of 3.9 days but no serious infection episodes occurred. Combined ifosfamide/epirubicin and standard G-CSF is able to mobilize sufficient PBPC without serious side-effects for patients with MM and for purging procedures resulting in a high proportion of complete remissions after tandem high-dose melphalan chemotherapy.

Lymphoma discrimination by computerized triple matrix analysis of list mode data from three-color flow cytometric immunophenotypes of bone marrow aspirates.

BACKGROUND: The goal of this study was to evaluate a self-learning algorithm for the computer classification of information extracted from flow cytometric immunophenotype list mode files from high-grade non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), and multiple myeloma (MM). Materials and Methods Bone marrow aspirates (BMA) were obtained from untreated NHL (n = 51), HD (n = 9), or MM (n = 13) patients. Bone marrow aspirates were not infiltrated in NHL and HD patients as confirmed by thorough histologic and cytologic investigation; however, MM patients showed an infiltration rate >50% by malignant myeloma cells. Peripheral blood leukocyte (PBL) samples were taken from age-matched healthy volunteers (n = 44) as easily available control material. A second control group of 15 healthy volunteers, from whom BMA and PBL samples were available, allowed us to differentiate whether the observed classification results on malignant samples were due to the malignant process or simply to the inherent differences between BMA and PBL. Bone marrow aspirates and PBL were analyzed by the same immunophenotyping antibody panel (CD45/14/20, CD4/8/3, kappa/CD19/5, lambda/CD19/5). The acquired list mode data files were analyzed and classified by the self-learning triple matrix classification algorithms CLASSIF1 following a priori separation of the data into a learning set and unknown test set. After completion of the learning phase, known patient samples were reclassified and unknown samples prospectively classified by the algorithm. RESULTS: Highly discriminatory information was extracted for the various lymphoma entities. The most discriminating information was encountered in antibody binding, antibody binding ratios, and relative antibody surface density parameters of leukocytes rather than in percentage frequencies of discrete leukocyte subpopulations. Samples from healthy controls were classified as normal in 97.2% of the cases, whereas those of NHL, HD, and MM patients were on average correctly classified in 80. 8% of the cases. CONCLUSIONS: Although no detectable lymphoma cells were present in BMA of NHL and HD patients, the CLASSIF1 classification of the immunophenotypes of morphologically normal cells provided a surprisingly good disease discrimination equal or better than that obtained by examining pathological lymph nodes according to the respective literature. The results are suggestive for a lymphoma-related and disease-specific antigen expression shift on normal hematopoietic bone marrow cells that can be used to discriminate the underlying disease (specificity of unspecific changes), i.e., in this case NHL from HD. Multiple myeloma patients were discriminated by changes on malignant as well as on normal bone marrow cells.

Comparative flow cytometric study of clonal excess in leukaemic peripheral blood from patients suffering from chronic lymphocytic leukaemia (B-CLL) by different antibodies, staining techniques and the effects of blood storage.

This investigation studied the effects of cell preparation methods, different antibody panels and blood storage on antigen expression of abnormal B lymphocytes from patients with B-CLL. Blood specimens collected in Heparin de novo were processed by using conventional Hypaque-Ficoll density gradient centrifugation and whole blood lysis. These were stored for 3 days at 4 degrees C, 24 degrees C and 30 degrees C. Although clonal excess was detected by all antibody panels, significant differences could be observed in terms of molecules of equivalent fluorochromes (MEF/MESF units). Evaluation of 'weak and strong' staining is dependent on the antibody panel used. Immunofluorescent values for CD19 and CD45 were unchanged at 4 degrees C and 24 degrees C but immunoglobulin staining showed best results when blood was stored at 4 degrees C. Storage at 30 degrees C produced unreliable results. Abnormal B lymphocytes should be analysed immediately after the specimen is obtained. If shipment is necessary they should be kept at 4 degrees C. Surface immunoglobulins are the 'antigens' most sensitive to storage alterations. Sample alterations alone are sufficient to the correct classification of NHL, especially in the case of low-grade NHL.

Enumeration of CD34-positive hematopoietic progenitor cells by flow cytometry: comparison of a volumetric assay and the ISHAGE gating strategy.

In order to optimize peripheral blood stem cell (PBSC) collection for transplantation, absolute CD34 counts are necessary to determine the exact time-point for sufficient leukapheresis. In an effort to establish and to validate a rapid and reproducible assay for PBSC enumeration, different recommendations for selection of monoclonal antibodies, lysis techniques, analysis parameters and gating strategies were developed. In this methodical study, two gating strategies for PBSC enumeration were compared, in order to validate the accuracy of PBSC counting in peripheral blood and apheresis products. Gating strategy I was performed using volumetric flow cytometry and reference beads whereas gating strategy II was done according to the ISHAGE guidelines. The highly standardized volumetric assay seems to be superior to the more 'expert-reliant' ISHAGE procedure requiring more 'manual work' by the cytometrist.

Prognostic significance of dysplastic features of hematopoiesis in patients with de novo acute myelogenous leukemia.

The detection of dysplastic features of hematopoiesis in de novo acute myeloid leukemia (AML) by light microscopy is defined as AML with trilineage myelodysplasia (AML/TLMD). The prognostic relevance of these dysplastic features for patients with de novo AML remains unclear. In order to evaluate the role of dysplasia in de novo AML, bone marrow aspirates from 69 patients were analyzed prospectively and investigated separately for erythropoiesis, granulopoiesis and megakaryopoiesis by three independent investigators. The overall complete remission (CR) rate was 48.8% and partial remission (PR) or nonresponders constituted 52.2% of the patients investigated. The median overall survival time was 5 months with a disease-free interval of 3.5 months for all patients. Dysgranulopoiesis (DysG) was observed in 30.4%, dysmegakaryopoiesis (DysM) in 50.7%, and dyserythropoiesis (DysE) in 43.5%. Of all patients, 26.0% showed trilineage dysplastic features and were thus classified as AML/TLMD. A significantly worse prognosis (Kaplan-Meyer plot, Student's t-test) was calculated for those patients with detection of only DysG (p = 0.002), DysM (p = 0.02), DysE (p = 0.04) as compared with patients without any dysplastic signs. An unfavorable karyotype was correlated with patients showing DysG (P = 0.02) and DysM (P = 0.04). For these patients with an unfavorable karyotype, the occurrence of any dysplastic features had no additional prognostic impact. Dysplastic features (DysG, DysM, DysE) seem to be an important prognostic factor in de novo AML correlating with short overall survival. DysG and DysM correlated well with the appearance of unfavorable chromosomal abnormalities. It may be reasonable to assume that patients with dysplastic features should be considered for more aggressive treatment schedules at the time of diagnosis.

Automated classification of patients with chronic lymphocytic leukemia and immunocytoma from flow cytometric three-color immunophenotypes.

The goal of this study was the discrimination between chronic lymphocytic leukemia (B-CLL), clinically more aggressive lymphoplasmocytoid immunocytoma (LP-IC) and other low-grade non-Hodgkin's lymphomas (NHL) of the B-cell type by automated analysis of flow cytometric immunophenotypes CD45/14/20, CD4/8/3, kappa/CD19/5, lambda/CD19/5 and CD10/23/19 from peripheral blood and bone marrow aspirate leukocytes using the multiparameter classification program CLASSIF1. The immunophenotype list mode files were exhaustively evaluated by combined lymphocyte, monocyte, and granulocyte (LMG) analysis. The results were introduced into databases and automatically classified in a standardized way. The resulting triple matrix classifiers are laboratory and instrument independent, error tolerant, and robust in the classification of unknown test samples. Practically 100% correct individual patient classification was achievable, and most manually unclassifiable patients were unambiguously classified. It is of interest that the single lambda/CD19/5 antibody triplet provided practically the same information as the full set of the five antibody triplets. This demonstrates that standardized classification can be used to optimize immunophenotype panels. On-line classification of test samples is accessible on the Internet: http://www.biochem.mpg.de/valet/leukaem1.html Immunophenotype panels are usually devised for the detection of the frequency of abnormal cell populations. As shown by computer classification, most the highly discriminant information is, however, not contained in percentage frequency values of cell populations, but rather in total antibody binding, antibody binding ratios, and relative antibody surface density parameters of various lymphocyte, monocyte, and granulocyte cell populations.

Differential effect of the activation of protein kinase A on the protein synthesis and secretion in the T-helper 2 cell line D10.G4.1.

The authors analysed the effect of protein kinase A (PKA) activation on the protein synthesis and secretion in the T-helper 2 cell line D10.G4.1 (D10) using an assay that allows the detection of almost all secreted proteins of a cell. IL-4 and IL-10 were quantified. Three groups of secretory products could be defined. The T-cell receptor (TCR)-induced production of the first group (A) of proteins including IL-4 was enhanced by low concentrations of PKA activators. At higher concentrations the enhancement was less marked. The synthesis and secretion of a second group (B) of proteins including IL-10 remained unaffected. The production of a third group (C) of proteins was inhibited in a concentration-dependent manner. Biochemical analysis revealed a block of phospholipase C gamma (PLC gamma) activity by PKA activators. When D10 cells were stimulated by a phorbol ester plus calcium ionophore the production of group A proteins was enhanced almost fourfold, whereas production of group B proteins was unaffected by PKA activation. This effect was observed at all concentrations of various PKA activators tested. The secretion of group C proteins was no longer inhibited. The same results were obtained when analysing IL-4 and IL-10 m-RNA by Northern blotting. The data demonstrate a lymphokine specific mode of action on a single cell basis. Furthermore, it suggests that the inhibitory action of PKA in D10 cells is due partly to blocking of PLC gamma activity.

BIRMA-K3, a new monoclonal antibody for CD34 immunophenotyping and stem and progenitor cell assay.

A murine monoclonal antibody with specificity for the CD34 antigen has been produced and designated BIRMA-K3. The antibody characterized as IgG1(kappa) has been shown to react with KG-1a cells following treatment of the cells with glycoprotease enzyme, indicating reactivity with the class III epitope of CD34. It was possible to show that class I and class II anti-CD34 antibodies were not able to inhibit binding of BIRMA-K3. Investigation of FITC-labeled as well as PE-labeled BIRMA-K3 resulted in a clear cut-off staining of acute leukemias and CD34+ cell counts in patients submitted to high-dose chemotherapy and stem cell transplantation. The results obtained correlate strongly with those from HPCA-2, the Becton-Dickinson class III antibody.

Infection by Alcaligenes xylosoxidans subsp. xylosoxidans in neutropenic patients.

Alcaligenes xylosoxidans subsp. xylosoxidans (A. x. xylosoxidans) is a nonfermenting gram-negative peritrichous rod and opportunistic pathogen. The organism is frequently found in an aqueous environment. In the past few years, nosocomial infections caused by A. x. xylosoxidans have become more evident. The literature suggests that systemic infections are severe and often lethal and an optimal antibiotic therapy is not well established. This report describes nosocomial infections in 11 patients of a hematology ward over a 2-month period. Primary infection occurred during the neutropenic phase after cytotoxic chemotherapy. Reinfection spread from central venous catheters that had been implanted before the first infection. The bacteremia was successfully treated by imipenem. None of the 11 patients died from the bacteremia, but 3 died of their underlying diseases. Despite an intensive search for the source, the route of infection remained uncertain. Nosocomial infections by A. x. xylosoxidans are of growing importance in high-risk patients. Although the source of infection often remains unknown, infection seems to originate from contaminated solutions. Treatment with imipenem and the removal of central venous catheter systems successfully eliminated A. x. xylosoxidans, which adheres to plastic material.

Lymphocyte gating of peripheral blood in patients with leukemic low-grade non-Hodgkin's lymphoma by multiparametric flow cytometry.

The standardized fluorescence intensity as expressed in molecules of equivalent soluble fluorescence (MESF) of lymphocytes from normal individuals and patients suffering from low-grade non-Hodgkin's-lymphomas was obtained comparing different staining patterns of CD45(FITC) and CD20(PerCP). After standardization of the flow cytometer using standardized fluorescent particles ('beads') significant differences could be obtained for hairy cell leukemia, chronic lymphocytic leukemia and immunocytomas in the peripheral blood. In contrast, centroblastic-centrocytic as well as centrocytic lymphomas showed no significant variations as compared to normal peripheral blood lymphocytes. According to these results, a new lymphocyte gating procedure was established by adding CD14(PE) and three-color measurement by CD45/CD14/CD20 staining of peripheral blood using erythrocyte lysis. The established gating procedure leads to a crucial discrimination and quantification of abnormal and normal lymphocytes per one measurement, whereas the 'leucogate' as defined by CD45/CD14 staining alone was insufficient for correct lymphocyte gate setting. In conclusion, the different staining of CD45 and CD20 in leukemic peripheral blood should be considered when fluorescence intensity or atypical peaks occurred in flow cytometric histograms suggesting for abnormal cell populations. In addition, it is possible to use this information to classify low-grade lymphomas.

Multiparametric immunophenotyping of B cells in peripheral blood of healthy adults by flow cytometry.

The investigation of patients suffering from malignant lymphomas of the B-cell type requires flow cytometric immunophenotyping. Several reports described the expression of almost all B lineage antigens on normal and abnormal B lymphocytes. Thus, immunophenotyping of lymphomas must be interpreted in the context of the reference values obtained for healthy control individuals. For this purpose multiparametric flow cytometric analysis offers the unique feature for lymphocyte subset analysis. In the present study B lymphocytes in the peripheral blood of healthy adults were investigated by multiparametric flow cytometric immunophenotyping for the detection of the frequency (in percent) of antigens provided by the revised European-American classification of lymphoid neoplasms (REAL) classification. Thus, 84 healthy adults were investigated and grouped by age (average ages were as follows: group 1, 25.38 years; group 2, 33.86 years; group 3, 44.17 years; group 4, 55.67 years; group 5, 66.67 years). Analysis was done for surface immunoglobulins (kappa and lambda chains of immunoglobulin M [IgM] and IgD) as well as CD10, CD11c, CD23, CD38, CD103, FMC-7, and B-B4. Three-color immunophenotyping was performed for kappa/CD19/CD5, lambda/CD19/CD5, surface IgM/surface IgD/CD19, FMC-7/CD19/CD5, CD103/CD11c/CD19, CD10/CD23/CD19, and CD38/B-B4/CD19 by live gating of CD19+ events (n = 2,000). Although some numerical differences could be obtained for the different groups, statistical differences (P < 0.005) could only be obtained for the CD19+/CD5+ B-cell subset, which was decreased in the elderly patients (group 5). The established two-color and three-color stainings will serve as a basis for future multiparametric immunophenotyping of abnormal lymphocytes (e.g., for patients suffering from non-Hodgkin's lymphoma of the B-cell type).

Immunophenotyping of B lymphocytes by multiparametric flow cytometry in bone marrow aspirates of healthy adults.

Establishing reference ranges by multiparametric immunophenotyping of mature B cells in bone marrow of healthy adults is of interest because the detection of bone marrow infiltration and persistence of light chain restriction, as well as discrimination between reactive and malignant lymphocytes are important applications of B-cell immunophenotyping. To determine the pattern of antigens as expressed by malignant mature B lymphocytes in the present study, bone marrow aspirates of healthy adults were investigated for the presence and percentage frequency of those antigens as defined for immunophenotyping of B cells by the REAL Classification. For this purpose, analysis of CD19-positive B lymphocytes by 'live gate' analysis was performed. The established two-color as well as three-color stainings will serve as a basis for future investigations designed to test multiparametric analysis of B lymphocytes in bone marrow aspirates. All investigated antibodies stained with varying percentage frequency on B-cell subtypes, and no statistical significant difference was found between bone marrow aspirates of women and those of men. On the basis of this analysis, all the reported lineage antigen combinations are present both in malignant B lymphocytes and in normal B cells in considerable percentage frequency. These findings are of importance for follow-up investigations of patients with non-Hodgkin's lymphomas by multiparametric immunophenotyping.

[Type I cryoglobulinemia]. / Typ-I-Kryoglobulinämie.

HISTORY AND CLINICAL FINDINGS: For 2 years a 52-year-old man had repeated bouts of purpura, arthralgia and fever. He was known to have abnormal monoclonal gammaglobulins, type IgG-lambda and vasculitis when he had another bout with acute renal failure and necrotizing ulcers in the legs. TESTS: Several laboratory tests were abnormal: erythrocyte sedimentation rate (122 mm), haemoglobin level (9.1 g/dl), white cell count (32,000/microliters), platelet count (562,000/microliters), creatinine level (4.1 mg/dl) and liver enzyme activities. He also had proteinuria (4.5 g daily) and nephritic urinary sediments. The immunoglobulin was subtype IgG3, and a cryoglobulinaemia was also present. Total complement level (CH 50) was not measurable. Bone marrow aspirate revealed plasmocytoma infiltration, and renal biopsy demonstrated necrotizing arteritis, as well as granular subendothelial deposits of IgG and complement. TREATMENT AND COURSE: After three plasma separations and initiation of the first treatment cycle with a four-day infusion of vincristine, doxorubicin and dexamethasone the creatinine concentration fell to within the normal range and the necroses healed slowly. No cryoglobulin activity has been demonstrable over the past 24 months.

Hyperviscosity syndrome: efficacy and comparison of plasma exchange by plasma separation and cascade filtration in patients with immunocytoma of Waldenström's type.

The hyperviscosity syndrome is a common problem in patients suffering from IgM paraproteinemia. In this situation cytotoxic chemotherapy alone is insufficient and additional plasma therapy is required. Until recently, conventional plasma exchange was the only plasma therapy available. While this method has proven its efficacy, it eliminates proteins unselectively. Cascade filtration, on the other hand, has been established to remove proteins as a function of their size offering the prospect of a highly selective withdrawal of macromolecules. In the work presented, the efficacy of conventional plasma exchange and cascade filtration was evaluated performing both techniques at random in cases of hyperviscosity syndrome due to immunocytoma of Waldenström's type (n = 11/group). In these patients, conventional plasma exchange decreased plasma viscosity by 48%; cascade filtration was less effective (26%), correlating with a smaller decrease of IgM (conventional plasma exchange 42% vs cascade filtration 27%). The profile of other plasma proteins studied did not change significantly with either treatment. Furthermore, we observed no differences regarding serious side-effects. In conclusion, we could not demonstrate a superior effect of cascade filtration as compared to conventional plasma exchange in the treatment of hyperviscosity syndrome.

Primary non-Hodgkin's lymphoma of the vagina. Case report and review of the literature.

The genital tract as primary site of malignant non-Hodgkin's lymphoma in women is extremely rare, whereas secondary involvement in advanced disease is found in about 40% of cases. In this report a patient is presented who had a primary vaginal non-Hodgkin's lymphoma of the centroblastic type according to the Kiel classification, with an excellent response to cytotoxic chemotherapy (CHOP) and event-free disease for 3 years. A review of the literature shows that favorable prognosis of localized disease seems to be a common experience. Primary involvement of the vagina can be successfully treated by pelvic irradiation, but in young women cytotoxic chemotherapy should be considered to preserve fertility.