Monocyte-mediated-antibody-dependent cellular cytotoxicity (MO-ADCC) was studied in 21 patients with Hodgkin's disease (HD), 15 patients with a long-lasting remission of HD, 11 patients with non-Hodgkin's lymphoma (NHL), 11 patients with solid tumors, and 15 normal controls. Lymphocyte ADCC (LY-ADCC) was evaluated in 12 patients with HD and 9 normal controls. Monocytes lymphocytes were isolated with cell-scatter monitored counterflow centrifugation providing high purity and yield. Antibody-dependent cellular cytotoxicity was evaluated by means of DNA flowcytometry, using antibody-coated chicken erythrocyte targets (CRBC). In comparison with normal controls MO-ADCC was significantly increased in HD (P less than 0.0005), NHL (P less than 0.005), and solid tumors (P less than 0.005). In patients in long-lasting complete remission of HD, MO-ADCC was in the normal range. Lymphocyte-ADCC of 12 patients with HD was similar to that of 9 normal controls. In all experiments LY-ADCC was invariably lower than MO-ADCC of the same donor, indicating the monocyte as the most potent effector cell towards CRBC targets. Results indicate the following: (1) purified cell suspensions of both lymphocytes and monocytes are essential to unravel their role as effector cells; (2) LY-ADCC in HD is similar to normal controls; (3) MO-ADCC enhancement is not uncommon in malignant lymphoma and several solid tumors; (4) normal MO-ADCC in a group of successfully treated patients with HD suggests a disease-related induction of enhanced MO-ADCC.

Adolescent↗

PubMed1984

Lymphocyte isolation from human spleen by counterflow centrifugation employing two different flow chambers on line.

Studies on splenic lymphocytes have hitherto been performed on single cell suspensions depleted of phagocytic cells by adherence to plastic or incubation with carbonyl iron. These techniques have the disadvantages of selective cell loss, suboptimal cell purification and cell activation. This paper describes purification of splenic lymphocytes by the use of counterflow centrifugation (CFC). The method was adapted to overcome pelleting of cells in the separation chamber to form a plug at the inlet and impede adequate flow. By combining 2 different separation chambers on line in 1 rotor this problem was overcome. Of all lymphocytes recovered after CFC 88.8 +/- 1.4% were collected in 2 pooled fractions with a purity of greater than or equal to 98% and a cell viability of 95%. After CFC, 80.8 +/- 12.1% of the viable cells loaded were recovered.

Blood Platelets↗

PubMed1984

Allogeneic bone marrow transplantation in a patient with acute myeloid leukemia secondary to Hodgkin's disease.

A patient with Hodgkin's disease entered complete clinical remission by combination radiochemotherapy. He developed dyshematopoiesis 1.5 years later and an overt acute nonlymphocytic leukemia 3 years after diagnosis. A complete remission was achieved following 2 courses of intensive polychemotherapy. Four months later, while still in remission, he underwent an allogeneic bone marrow transplantation (BMT) from an HLA-identical sister. Mild chronic graft versus host disease of the skin occurred 3 months after BMT, and now the patient has been in complete remission of leukemia for over 2 years. This appears to be a unique case of prolonged remission of a leukemia secondary to an intensively treated Hodgkin's disease.

Acute Disease↗

PubMed1984

Calculation of S-phase numbers of four major cell categories in human bone marrow from DNA-flow cytometry and counterflow centrifugation data.

Fractionation of heterogeneous cell populations into a number of fractions differing in cell size and composition, followed by measurement of DNA profiles and differential morphology of the samples allows the calculation, using a mathematical deconvolution procedure, of the cell proliferation of the individual categories. The method was applied to human bone marrow, fractionated by counterflow centrifugation into 10 to 15 fractions. Calculated percentage of S-phase cells in four major categories, including myeloid, erythroid, monocytic, and lymphoid cells, were in good agreement with data obtained by tritiated thymidine autoradiography.

Bone Marrow↗

PubMed1984

An easy-to-build-timer for kinetic measurements in flow cytometry.

Measuring kinetics of any time-dependent intracellular process on a cell-to-cell basis is a powerful extension of flow cytometry (FCM) possibilities. A digital timer is described that is easy to build and that fits with all commercial or custom made multiparameter equipment. Of all possible applications of this timer (phagocytosis kinetics, staining of cell components, enzyme reactions, etc.), one example is given: enzymatic intracellular precipitation owing to myeloperoxidase in human peripheral blood cells.

Flow Cytometry↗

PubMed1984

Separation of immunoreactive lymphocytes from human pluripotent stem cells (CFU-GEMM) by means of counterflow centrifugation.

Counterflow centrifugation with continuous monitoring of the output for cell number and cell scatter was used to separate low density (d less than 1.070 g/ml) human bone marrow cells in two fractions: one containing the majority of small size lymphocytes and the other the majority of the larger sized committed progenitor cells. The recovery of the pluripotent stem cells (CFU-GEMM) in the large cell fraction was complete. The mitogenic reactivity of this putative stem cell fraction had decreased to 6% and 11%, of the original value as measured with phytohemagglutinin stimulation and one way mixed lymphocytic culture respectively. Counterflow centrifugation offers a physical separation technique, by which the majority of the immunoreactive cells can be separated from the pluripotent hematopoietic stem cells.

Bone Marrow Cells↗

PubMed1984

Combined immunodeficiency preceding chronic lymphocytic leukemia.

A 66-year-old female, suffering from recurrent infections of the respiratory tract, developed a severe hypogammaglobulinemia and an impaired cell mediated immunity in combination with abdominal lymphadenopathy and splenomegaly. Considering a lymphoid malignancy, an explorative laparotomy was performed. No malignancy could be established. After a follow-up of 2 years a diagnosis of chronic lymphocytic leukemia could be made. The onset of hypogammaglobulinemia 2 years before the development of chronic lymphocytic leukemia is not yet reported in the literature.

Agammaglobulinemia↗

PubMed1984

Clinical trial of low-dose Ara-C in the treatment of acute leukemia and myelodysplasia.

In a multicenter analysis, the effect of low-dose cytosine arabinoside (Ara-C)(10 mg/ m2q 12 h subcutaneously for a minimum of 15 days) has been assessed in 13 patients with acute leukemia (10 myeloid-AML-, 3 lymphocytic-ALL-) and 7 patients with dysmyelopoietic syndromes (DMPS), conditions classified as refractory anemia with an excess of blasts ( RAEB ). Seven patients suffering from acute leukemia and 1 with DMPS in blastic transformation displayed a leukocytosis of more than 10 X 10(9)/1. Three out of 7 DMPS, 1 out of 10 AML achieved a complete remission, 1 out of 3 ALL-patients reached a partial remission twice. Seven patients showed a blast clearing in the bone marrow and peripheral blood, in another 7 instances examination of the bone marrow was not performed after therapy because of early death. The majority of patients were in their late phase of disease and refractory to conventional chemotherapy. Only 5 patients had no pretreatment at first presentation before low-dose Ara-C was initiated. At least for the DMPS-group, this therapeutic approach seems to be of some benefit.

Anemia, Aplastic↗

PubMed1984

Bone marrow repopulation capacity after transplantation of lymphocyte-depleted allogeneic bone marrow using counterflow centrifugation.

Bone marrow from six allogeneic HLA-matched and MCL nonreactive siblings was fractionated by means of isopycnic flotation centrifugation and subsequent counterflow centrifugation. The low density fraction (d less than or equal to 1.070 g/ml) obtained by IFC contained 20% of the nucleated cells and more than 90% of the myeloid and erythroid progenitors. The putative stem cell fraction obtained by CC showed a satisfactory recovery (88%) of the CFU-GM and BFU-E and only 3.5% of the original number of T lymphocytes. Bone marrow repopulation capacity was not impaired in comparison with a comparable group of patients. Despite the average high age of this group (29.6 years), only one of the four evaluable patients developed graft-versus-host disease.

Adolescent↗

PubMed1984

Separation of human bone marrow by counterflow centrifugation monitored by DNA-flowcytometry.

Human bone marrow was fractionated by counterflow centrifugation into 16 fractions with increasing cell size. Three distinct subpopulations could be recognized: small lymphocytic cells, medium-sized nucleated erythroid cells and large myeloid elements. DNA-flowcytometry and 3H-thymidine uptake showed that within the erythroid and myeloid cell populations counterflow centrifugation separates each population according to the cell cycle phase. Hypotonic treatment of bone marrow for removal of the erythroid nucleated cells resulted in a complete abrogation of the proliferating erythroid cell population. Counterflow centrifugation also separates the small non-proliferating myeloid and erythroid committed stem cells from the larger proliferating stem cells. It appeared feasible to separate the small lymphocytic cells from the majority of BFU-E and CFU-GM, due to the larger size of the proliferating normoblasts and the committed progenitor cells. Elimination of the mature lymphocytes from the haematopoietic stem cells by counterflow centrifugation may offer an alternative approach to the prevention of graft versus host disease (GvHD).

Bone Marrow↗

PubMed1984

Clinical course and survival in 16 patients with localized plasmacytoma.

The clinical course of 16 patients with localized plasmacytoma, 9 with solitary plasmacytoma of bone (SPB), and 7 with extramedullary plasmacytoma (EMP) are presented. Median follow up of SPB was 77 months and of EMP 74 months. The EMPs were localized in the upper respiratory tract (4 cases), the gastro-intestinal tract (2 cases) and the skin (1 case). The SPBs were localized in the ribs (3 cases), the spine (3 cases), the humoral bone (1 case), the skull (1 case) and the mandibular bone (1 case). In 3 patients with EMP and in 5 patients with SPB, the monoclonal protein could be determined at presentation. Local recurrences following surgical removal or irradiation occurred in 2 patients with EMP and in 1 patient with SPB after 12-60 months. Classical multiple myeloma developed in 3 patients with EMP and in 4 patients with SPB, 9-130 months after diagnosis. The monoclonal protein level proved to be a useful disease marker which (re)appeared at local recurrence or at dissemination. The median survival of the 16 patients with solitary plasmacytoma was 138 months, which is considerably longer than the median survival of 42 months in 14 stage-I patients with classical multiple myeloma who were observed during the same follow-up period.

Adult↗

PubMed1983

Cell size monitored counterflow centrifugation of human bone marrow resulting in clonogenic cell fractions substantially depleted of small lymphocytes.

Human bone marrow cells were fractionated by physical methods in order to obtain cell fractions enriched in clonogenic cells and devoid of immunocompetent lymphocytes. The bulk of the erythrocytes was removed by isopycnic gradient centrifugation on Ficoll-Isopaque (d = 1.085 g/ml) and the majority of mature granulocytes on Percoll (d = 1.070 g/ml). The nucleated cells were separated into fractions by counterflow centrifugation. Continuous monitoring of the effluent of the elutriator by a light scatter device improved the reproducibility of the separation profiles. Progenitor cells did not form a single distinct peak and the maximal enrichment factor was 8.5. Lymphocytes were eliminated almost completely from the progenitor cell rich fraction (both CFU-GM and BFU-E). Physical elimination of lymphocytes from human bone marrow may offer an alternative approach to the prevention of graft-versus-host disease in allogeneic bone marrow transplantation.

Antigens, Surface↗