Evaluation of doxorubicin in combination with mafosfamide for in vitro elimination of myeloid and lymphoid tumor cells from human bone marrow.

In an attempt to improve in vitro pharmacological purging of autologous grafts, the ability of doxorubicin (DOX), alone and in combination with mafosfamide (AZ), to eliminate tumor cells from human bone marrow was assessed. HL60 and Raji cells were mixed with a 20-fold excess of normal marrow cells and were incubated either with DOX (0.4-3.2 micrograms/ml) for 1 h or AZ (20-140 micrograms/ml) for 30 min or both drugs sequentially. Cytotoxicity was evaluated on tumor cells and GM-CFU by clonogenic assays and on earlier hemopoietic progenitors by liquid long-term marrow cultures (LTMC) for 5 weeks. DOX at 3.2 micrograms/ml and AZ at 140 micrograms/ml spared 1.08 and 1.23% of GM-CFU respectively, and yielded similar tumor cell log-kills for HL60 cells (3.04 and 2.95) and Raji cells (3.24 and 3.40). With the combination of AZ and DOX, the best therapeutic index was observed when the cells were incubated with AZ prior to DOX. Under these conditions, AZ at 80 micrograms/ml together with DOX at 1.6 micrograms/ml significantly increased log-kill values for HL60 cells to 3.96 by a synergistic effect and for Raji cells to 3.85 by an additive effect. In LTMC, GM-CFU recovery after treatment with AZ alone and with the combination of AZ and DOX was 59.9 and 20.0%, respectively, while it was 7.9 and 2.9% at culture initiation. These results suggest that the purging efficiency of DOX is comparable to that of AZ and may be enhanced by combination with AZ.

Erythrocytes as carriers for L-asparaginase. Methodological and mouse in-vivo studies.

L-Asparaginase has been encapsulated in Swiss mouse or human erythrocytes by hypotonic haemolysis followed by isotonic resealing and reannealing. The details of incorporation and properties of carrier erythrocytes are presented. When L-asparaginase loaded into 51Cr-labelled erythrocytes, was infused intravenously, the same half-life was found for asparaginase and 51Cr. In addition, L-asparaginase loaded into erythrocytes was much more effective in eliminating plasma asparagine compared with the same dose of free L-asparaginase injected in solution, during a sustained period (14 days).

Engineered erythrocytes: influence of P50 rightward shift and oxemia on oxygen transport to tissues.

The red blood cell (RBC) membrane may be reversibly opened using a lysis-resealing continuous flow method. The technology was adapted to the internalisation of an allosteric effector of haemoglobin, Inositol-Hexaphosphate (IHP). This molecule, occupying the allosteric site of 2,3 Bis-Phosphoglycerate with a very large affinity, induces a rightward shift of the oxyhaemoglobin dissociation curve (ODC). From ODC parameters in human volunteers, the potential effect of P50 (oxygen pressure at 50% haemoglobin saturation) on oxygen exchangeable fraction (OEF%), for various oxygen partial pressures (oxemia) was evaluated. For hyperoxic or normoxic arterial oxygen pressure (paO2), rightward shift greatly improved OEF%. In optimised conditions, engineered erythrocytes were potentially able to deliver two to three times more oxygen than normal cells. For patients with decreased paO2, as observed in chronic obstructive pulmonary deficiency (COPD), the reduction in arterial oxygen saturation (saO2%) reduces the benefit of the treatment for paO2 values between 60 and 80 mmHg. Below 60 mmHg, the saO2% reduction cannot be compensated by a corresponding reduction in svO2%, particularly for organs with physiologically low svO2%. In these organs, deleterious effects could be observed for a very large rightward shift of the ODC. Such engineered cells have unique properties for oxygen transport improvement and may be used for the treatment of patients suffering from diseases associated with hypoxia and ischemia.

Internalization and distribution of inositol hexakisphosphate in red blood cells.

Inositol hexakisphosphate (InsP6), an allosteric effector of haemoglobin, is able to modify the oxyhaemoglobin dissociation curve. The rightwards shift of the curve increases the in vivo oxygen delivery to tissues. Such an exogenous substance may be internalized into red blood cells (RBC) using a reversed lysis-resealing process following a hypoosmotic shock, resulting in InsP6-RBC with modified oxygen transport capacity. The efficacy of the process depends on various physicochemical parameters which can be fixed during the experimental protocol. The variability of InsP6 internalization from one sample to another appeared to be mainly due to the natural variation in osmotic fragility of RBC. This factor was also modified during the storage of RBC units before the lysis-resealing process. The separation of InsP6-RBC on a density gradient revealed a wide heterogeneity of internalized InsP6 concentration, varying with the degree of osmotic shock. The control of these various parameters will result in resealed InsP6-RBC in reproducible conditions suitable for in vivo use.

[Quantitative and thermodynamic study of B antigen in a family including individuals with phenotypes B 3 and AB 3]. / Etude quantitative et thermodynamique de l'antigène B dans une famille comportant des sujets de phénotype B3 et AB3.

Four B3 and one A1B3 erythrocyte samples belonging to the same family were studied using several series of quantitative measurements: percentage of agglutination, agglutination kinetics and thermodynamic methods. For this last assay the erythrocytes were used fresh and after treatment by formalin. From the obtained results evidence was given that while B antigen density was lower in A1B3 than in B3O cells, the reactive structure is qualitatively the same in these two kinds of cells. The enthalpy change of the reaction of this B3 antigen with a non stimulated anti-B from A1O individual was -- 5,000 cal/mole, i.e. weaker than when normal B cells were concerned (-- 16,000 cal/mole).

Quality, ISO 9002 and haemapheresis service.

Hemotherapy in developed countries is safer than ever but the present perception of society seems different than in the past. As Isbister wrote, during recent years, as blood safety has been progressively improved, the legal risks have been increasing. The first task is to study and define the risks for each activity. For me the management of risks may be achieved through the management of quality. The confidence of society in blood transfusion can probably best be obtained through a kind of recognized external quality assurance. I intend to describe our targets, our difficulties and the factors that have made our certification a success. I shall focus on several points: what quality represents; what our haemapheresis service is; what ISO 9002 standards are and why we choose them; our difficulties and the factors of success.

[HLA B27 phenotyping using flow cytometry]. / Phénotypage HLA B27 par cytométrie de flux.

Results of typing for HLA B27 antigens of 106 patients in whole blood with a FITC monoclonal anti HLA B27 antibody and analysis by flow cytometry are compared to the results obtained by the classical microlymphocytoxicity test. By flow cytometry analysis, there are not false negative result, but, because of cross reactions of the used monoclonal antibody with other specificities of the CREG B7 group, false positive result may be encountered. The flow cytometry analysis for typing HLA B27 antigens using a monoclonal antibody can be a good screening technique: so it's rapid and moreover the negative results can be accepted. The positive results must be confirmed.

Several aspects of red blood cell engineering: potential therapeutic applications.

Erythrocytes can be used to entrap drugs, enzymes or other molecules with active properties, with various encapsulation procedures. The method of internalization we are using includes an hypotonic dialysis step. Carrier erythrocytes survival depends on the dialysis process and the carried molecule. Research has led us to perform preclinical trials on animals for several drugs and enzyme therapies and for the improvement of oxyphoric capacity of erythrocytes. There exist many potential clinical applications for each kind of internalized molecules.

[Radioimmunoassay of antihemophilic factor A]. / Dosage radioimmunologique du facteur antihémophilique A

A radioimmunological method for the estimation of anti-haemophilic factor A was perfected and studied, making it possible to measure factor VII antigen as distinct from procoagulant factor VIII activity. The method uses a rabbit anti-factor VIII antibody, purified and labelled with I125. The technique of estimation is based upon the differential precipitability of specific factor VIII antigen-antibody complexes, which precipitate with 25 per cent saturated ammonium sulphate and of free antibody which does not precipitate under these conditions. The method is reproducible and sensitive, with the possibility of measuring 0.3 per cent of factor VIII antigen, i.e. 0.003 units/ml. Its specificity is confirmed by the absence of factor VIII antigen in a patient suffering from a severe form of von Willebrand's disease. Using this technique, normal subjects were found to have 96 +/- 31 per cent of factor VIII antigen in comparison with a pool of 25 normal plasmas. Haemophiliacs tested had, as would have been expected, antigen levels close to normal.

[In vitro study of platelet preservation during 5 days in reduced-thickness bags]. / Etude in vitro de la conservation de plaquettes pendant 5 jours dans des poches d'épaisseur réduite.

An in vitro study of platelet concentrates storage for 5 days was performed in PVC bags. Modification of the original three-day containers were introduced by thickness reduction. The results at fifth day were comparable to those at third day in standard plastic bags. During storage, variations of platelet counts were very slight with a low LDH release. PH was stable with a good maintenance of phase microscope platelet morphology. PCO2 and PO2 measurements showed a satisfactory gas permeability which could explain a limited lactate production. If in vivo studies of transfusion recovery confirm these data, platelet concentrates storage could be extended up to 5 days by such modifications of standard three-day PVC bags.

[Cad antigen in the French population]. / L'antigène Cad dans la population française

An investigation of Cad phenotypes in the French population had been carried out in 1973, in four Blood Transfusion Centers (Mulhouse, Nancy, Paris, Versailles), B and O red cells were tested with the Dolichos Biflorus lectin. Out of 78.528 donors, 56 were found to have the Cad antigen on their red cells. The mean frequency was 0,07%. Nevertheless, this frequency varied among the four above mentioned Blood Transfusion Centers: the observed differences were probably due to the preparation procedure of the Dolichos biflorus extract. The family investigation permitted the analysis of four families with at least three Cad individuals. The independence of the Cad system and of Auberger, Gc, Hp, C'3, PGM, Pac and ADA was demonstrated. A quantitative agglutination study on these Cad samples using the Dolichos biflorus lectin,and a selected AB serum showed a high variability of the erythrocyte Cad Strength, even within one family. Most Cad samples were found polyagglutinable when a sensitive technique and selected AB sera were used. All adult sera contained an anti-Cad1 antibody, except Cad1 individuals. Although strong Sda reactivity was always found in Cad red cells, the anti-Cad and anti Sda specificities were not identical: this was demonstrated by the absorption and inhibition tests of anti-Cad and anti-Sda reagents absorption and inhibition tests of anti-Cad and anti-Sda reagents with Sda material. From thf Cad red cells, there was no evidence of the existence of separable anti-A1 and anti-Cad agglutinins in the Dolichos biflorus lectin.

Thrombocytopenia in systemic lupus erythematosus: association with antiplatelet and anticardiolipin antibodies.

Serum platelet bindable immunoglobulin G (SPbIgG) and anticardiolipin antibodies (ACA) have been assayed in SLE patients with (group A, n = 8) and without thrombocytopenia (group B, n = 8). Moreover, we studied the binding of serum IgG on platelet proteins using a Western blotting procedure. Increased levels of SPbIgG were found in all thrombocytopenic patients and in five cases of group B. A binding of serum IgG on platelet proteins (80 and 53 kDa) was seen in seven of eight thrombocytopenic patients and in only one case of group B. ACA were present in the serum of four patients in each group and a significant inhibition of ACA IgG by entire washed platelets was achieved in only two severe thrombocytopenic cases. These data suggest that participation of ACA to the platelet destruction can be evoked for a few SLE patients, whereas recognition of platelet protein determinants by lupus autoantibodies is necessary to the thrombocytopenia.