Nonlabeled secondary antibodies augment/maintain the binding of primary, specific antibodies to cell membrane antigens.

BACKGROUND: In studies on surface membrane antigen expression using immunofluorescence techniques, it is commonly observed that direct staining gives weaker signals than the signals following indirect staining with fluorochrome-conjugated secondary antibodies. This is most marked when cells have also been permeabilized in order to stain intracellular protein. The commonly accepted explanation for this observation is that fluorochrome-conjugated secondary antibodies bind to a higher number of binding sites on the primary antibody, as compared to the binding of conjugated primary antibodies to the membrane antigens. Another hypothesis might be that the antibody/antibody complexes formed on the membranes when using the indirect technique may have an augmented ability to bind the membrane epitopes. The present study was performed in order to check this hypothesis. MATERIALS AND METHODS: Peripheral blood mononuclear cells were stained with fluorochrome-conjugated anti-CD antibodies directly without or with a second-step application of nonconjugated goat anti-mouse IgG antibodies, followed by different fixation and permeabilization methods. The cells were analyzed by flow cytometry. RESULTS: A second-step application of nonconjugated goat anti-mouse IgG antibodies following direct staining with fluorochrome-conjugated anti-CD antibodies gave a significant increase in membrane antigen expression on permeabilized cells as compared to direct staining alone. The secondary antibody must be bivalent, since whole IgG or F(ab')(2) fragments of the goat anti-mouse antibodies showed effects, while Fab fragments did not. CONCLUSIONS: Nonlabeled secondary antibodies are able to influence the binding of primary, specific antibodies to cell membrane antigens on cells treated with permeabilizing agents necessary for staining intracellular proteins. The improved membrane antigen expression seems to be due to the formation of a network of primary and secondary antibodies on the cell surface, with increased ability for maintaining binding to CD antigens.

Health-related quality of life in multiple myeloma patients receiving high-dose chemotherapy with autologous blood stem-cell support.

In a population-based study, the Nordic Myeloma Study Group found a survival advantage for high-dose melphalan with autologous blood stem-cell support compared to conventional chemotherapy in myeloma patients under 60 yr of age (risk ratio: 1.62; confidence interval [CI] 1.22-2.15; p = 0.001). A study of health-related quality of life (HRQoL) was integrated in the trial, using the EORTC QLQ-C30 questionnaire. Of the 274 patients receiving intensive therapy 221 (81%) were compared to 113 (94%) of 120 patients receiving conventional melphalan-prednisone treatment. Prior to treatment, there were no statistically significant differences in any HRQoL score between the two groups. One month after the start of induction chemotherapy, the patients on intensive treatment had more sleep disturbance than the control patients. At 6 mo, corresponding to a mean of 52 d after high-dose melphalan, the patients on intensive treatment had moderately lower scores for global QoL and role and social functioning and there was also a significantly higher score for appetite loss. At 12 and 24 mo, the HRQoL was similar to that of the control patients. At 36 mo, there was a trend toward less fatigue, pain, nausea, and appetite loss in the intensive-treatment group. Thus, the 18 mo of prolonged survival seem to be associated with a good health-related quality of life. Despite the moderate HRQoL reduction associated with the early intensive chemotherapy phase, this treatment modality must be regarded as an important step forward in the care of multiple myeloma.

Impact on survival of high-dose therapy with autologous stem cell support in patients younger than 60 years with newly diagnosed multiple myeloma: a population-based study. Nordic Myeloma Study Group.

High-dose therapy has become a common treatment for myeloma. The objectives of this study were to estimate in a prospective, population-based setting the impact on survival of high-dose therapy in newly diagnosed, symptomatic patients less than 60 years old and to compare the results with those of conventionally treated historic controls. The prospective population comprised 348 patients. Of these, 274 were treated according to a specified intensive-therapy protocol (Nordic Myeloma Study Group [NMSG] #5/94) and constituted the intensive-therapy group. The historic population consisted of 313 patients identified from 5 previous population-based Nordic studies. Of these, 274 fulfilled the eligibility criteria for high-dose therapy stated in NMSG #5/94 and constituted the control group. The expected numbers of patients in the prospective population and the historic population were 450 and 410, respectively, estimated from previously established data on the incidence in this population and the population base for each study. Survival was prolonged in the intensive-therapy group compared with the control group (risk ratio for the control group 1.62; 95% confidence interval 1.22-2.15; P =.001). These groups represented more than 60% of the expected number of patients. When survival for all the registered patients in the 2 populations was compared, representing more than 75% of the expected number of patients, the advantage for the prospective population persisted (risk ratio for the historic population 1.46; 95% confidence interval 1.14-1.86; P =. 002). These results indicate that the introduction of high-dose therapy for newly diagnosed myeloma has resulted in prolonged survival for the total patient population aged less than 60 years. (Blood. 2000; 95:7-11)

[Chronic myeloid leukemia in health regions 1, 3, 4 and 5 during the period 1990-96]. / Kronisk myelogen leukemi i helseregion 1, 3, 4 og 5 i perioden 1990-96.

The aim of the present investigation was to obtain information about treatment, clinical course and outcome for all patients with chronic myeloid leukaemia through a six-year period in a defined part of Norway. A total number of 141 patients fulfilled the diagnostic criteria. This is equivalent to 0.9 patients per 100,000 per year. The median age was 62 years. More than 70% of the patients were primarily treated with hydroxyurea, either alone or combined with interferon. 40 out of 57 patients younger than 55 years underwent allogeneic stem cell transplantation. Median survival for all patients was 36 months with an estimated five-year survival rate of 33%. Patients older than 55 years had a median survival of 30 months with 16% alive after five years. The five-year survival rate for patients younger than 55 years was 56%, for transplanted patients 72%. 60 of 84 patients older than 55 years have died after 4 1/2 years median observation time. Two thirds of those died of leukaemia; one third of other causes. 23 of 57 patients younger than 55 years have died. 11 of them had had transplantations and most of them died from transplantation-related causes, while leukaemia was the dominating cause of death in the others.

Elevated serum concentrations of hepatocyte growth factor in acute myelocytic leukaemia.

Serum concentrations of hepatocyte growth factor (HGF) were measured in 60 patients suffering from acute myelocytic leukaemia (AML). At the time of diagnosis elevated HGF concentrations (> 1.25 ng/ml) were found in 28% of the patients. HGF levels correlated with the presence of disseminated intravascular coagulation (DIC), levels of lysozyme, creatinine, peripheral blood blast counts and lactic dehydrogenase. In the group of patients with high HGF (>1.25 ng/ml) we found a tendency towards an increased early mortality; 41% of them died within 15 d from diagnosis, as opposed to 5% of the patients with normal HGF (log rank test p=0.07). DIC-related bleeding or thrombosis contributed to this early mortality. In responders, HGF levels normalized after treatment. HGF levels are low in neutropenia and neutropenic infections.

Survival in 86 patients, aged 15-60, with primary acute myelogenous leukemia, treated with a common program in the Norwegian health regions I, III, IV and V in the period 1990-1995.

Eighty-six patients between 15 and 60 yr with primary acute myelogenous leukemia in health regions I, III, IV and V in Norway were treated according to a common protocol from 21 January 1990 until 1 September 1995 (until 1 January 1993 for health region IV). Seventy-one percent of the patients reached complete remission (CR) and went on to receive consolidation treatment. In addition to chemotherapy, 18 patients under the age of 52, i.e. 28% of all patients in this age group, received allogeneic bone marrow transplantation. A follow-up analysis was performed by 1 September 1996. The median overall survival was 15 months, estimated 3-yr survival 30% and estimated survival at 5 yr was 26%. The median duration of 1st CR was 19 months, and the leukemia-free survival at 3 yr was 29%. At follow-up 26/86 patients were alive, 18 in 1st CR (4 after BMT) and 8 in 2nd CR (5 after BMT in 2nd, 1 after BMT in early 1st relapse). These results are comparable to many previously published studies, but may be inferior to the results obtained with more intensive consolidation treatment, including high dose Ara C.

[Treatment with growth factors and cytokines in hematologic diseases]. / Behandling med vekstfaktorer og cytokiner ved hematologiske lidelser.

The Norwegian Society of Haematology has worked out guidelines for the use of granulocyte-colony stimulating factor and granulocyte-monocyte colony stimulating factor and interferon alpha in clinical haematological practice. We recommend not using growth factors as a routine to prevent or to treat fever in patients with granulocytopenia induced by cytostatics, or patients with myelodysplastic syndromes. At present such treatment should be restricted to clinical trials. The same conclusion was reached in regard to use of erythropoietin in the case of myelodysplastic syndromes. Harvesting of stem cells from peripheral blood is a well documented indication for administration of growth factors. Interferon alpha as maintenance treatment for cases of multiple myeloma and low grade malignant lymphoma delays progression of the disease but does not improve chance of survival. There is no documentation of improved quality of life. Use of interferon alpha is not justified as a routine treatment for multiple myeloma. In chronic myelogenous leukemia, interferon alpha seems to be equal to or better than hydroxyurea, and may be considered for patients who cannot undergo allogeneic bone marrow transplantation.

Acute leukemia in middle Norway 1984-88.

One hundred and eight adult patients with acute leukemia were diagnosed in the middle Norwegian health region during the 5-year period 1984-88, giving an incidence rate of 4.6/100,000 per year. Nine patients had acute lymphoblastic leukemia (ALL), 93 acute myeloid leukemia (AML) and 6 patients acute leukemia without definite sub-classification. The median age of AML patients was 66 years. Thirty-five patients (median age 78 years) were found non-suitable for cytotoxic drugs, while 58 AML patients (median age 57 years) were given aplasia-inducing drug combinations according to one of three treatment programs depending on the time of diagnosis and age, in order to induce remission. Six patients were given oral drugs or low dose ara-C. All patients were followed until death or for an observation time of more than 5 years (median 7 years). The overall long term survival was found to be 12/108 for all acute leukemias, 8/93 for AML patients and 4/9 for ALL patients. For the AML patients given intravenous aplasia-inducing drugs the remission rate was 0.65, the median remission duration 12.2 months and the 5-year survival rate 0.19. For 31 AML patients, (median age 41 years), started on an intensive chemotherapy program, the 5-year survival rate was 0.32 and the relapse-free 5-year survival rate for the 22 patients entering complete remission was also 0.32.