Immunologic consequences of blood transfusion and their clinical manifestations.

Aspects of clinical immunology, in the context of transfusion medicine, became highlighted in the last decade as a consequence of the accelerating expansion of basic immunology, immunogenetics and molecular biology. In addition sophisticated new technologies, which were capable of producing pure and safe blood products, attracted more attention to research and monitor the consequences of transfusion. These technologies also had obvious effects on supportive hematological therapy. The transfusion of blood components follows the rules of organ transplantation: when there is a mismatch between the donor and the recipient, the transfusion has the potential to induce various kinds of immune response against alloantigens. Antigen-compatible transfusions that involve major and rare blood groups are in almost all cases mismatched with respect to various polymorphic systems expressed on the cellular blood components. These include histocompatibility leukocyte antigens (HLA), tissue-specific and differentiation alloantigens, and, in the case of plasma, immunoglobulins, complement components, heat shock proteins, and shedded soluble membrane alloantigens. Clinical manifestations of alloimmune responses are typically deleterious. For example, immediate antigen-antibody binding and its consequences as secondary activations are paralleled by the nonhaemolytic febrile reaction, HLA sensitization can lead to a state of platelet refractoriness and inconvenient clinical symptoms. In certain immunogenetic situations and in immunodeficient patients graft-versus-host disease can be induced by blood products that contain live lymphocytes. Leukocyte filtration techniques are widely used to avoid most but not all of these harmful side effects of blood component therapy. In contrast to these harmful side effects in certain immunogenetic conditions, alloantigens that are expressed on various blood products can elicit an advantageous suppression of the immune response in the recipient. In the context of kidney transplantation this is termed the 'beneficial transfusion effect', and typically results in the prolongation of the graft's survival. In cases of recurrent habitual abortion and IgG therapy associated with certain autoimmune diseases, immunization with leukocytes specifically takes advantage of this phenomenon. To date the beneficial transfusion effect is not fully understood. In certain cases of malignancies or gastrointestinal surgeries this suppression of immune regulation that is induced by transfusion can worsen the clinical state either by permitting the spread of the tumor or by allowing severe infections to proceed unchecked. In conclusion it is imperative to monitor the immunological consequences of transfusion in order to deter the disadvantageous side effects. Taking advantage of the 'beneficial transfusion effect' may also provide a new means for immune therapy using the various blood products.

Search for the pathogenesis of the differing phenotype in two compound heterozygote Hungarian brothers with the same genotypic triosephosphate isomerase deficiency.

In a Hungarian family with triosephosphate isomerase (TPI) deficiency, two compound heterozygote brothers were found with the same severe decrease in TPI activity, but only one of them had the classical symptoms. In search for the pathogenesis of the differing phenotype of the same genotypic TPI deficiency, an increase in red cell membrane fluidity was found. There were roughly 100% and 30% more 16:0/20:4 and 18:0/20:4 diacyl-phosphatidylcholine species in erythrocytes from the two TPI-deficient brothers than in the probes from healthy controls. The activities of acethylcholinesterase and calmodulin induced Ca2+ ATPase were significantly enhanced in erythrocytes from the propositus as compared with those of the neurologically symptom-free brother and other members of the TPI-deficient family as well as to those from healthy controls. Both enzymes are crucially involved in the function of nerve cells. The observed differences in membrane fluidity and enzyme activities between the erythrocytes from the phenotypically differing TPI-deficient brothers underline the importance of investigations into the effect of biophysical changes in the lipid environment of the membrane proteins on the development of disseminated focal neurological disorders of unknown pathogenic origin.

Blast colony forming cells in umbilical cord blood: frequency and correlation to other haemopoietic progenitors.

Umbilical cord blood has been suggested as a potential source of haemopoietic stem cells for clinical transplantation. Assay of stroma adherent blast colony forming cells (CFU-BL), as a cell type that may predict marrow repopulation, has already been suggested to predict the outcome of bone marrow transplantation. In the present experiments the frequency of CFU-BL in plastic non adherent mononuclear cell fraction (PNAMNC) of umbilical cord blood was studied. If PNAMNC from cord blood was co-incubated with a pre-established stromal layer from normal bone marrow a strict linear correlation between panned PNAMNC and blast colonies were observed. Frequency of CFU-BL in cord blood was significantly lower than that in bone marrow. In cord blood, CFU-GEMM frequency was found to be comparable to that of bone marrow, CFU-GM and BFU-E frequency was reduced. A good correlation between CFU-BL and BFU-E frequency was found both in cord blood and bone marrow. On the other hand, irrespective of their source, correlation between CFU-BL and CFU-GEMM or CFU-GM was weak.

Erythrocyte lipids in triose-phosphate isomerase deficiency.

Marked hypoalphalipoproteinemia was found together with relatively low serum cholesterol, triacylglycerol, and LDL levels in a triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1)-deficient Hungarian family, especially in the two compound-heterozygote brothers. Apart from a slight increase in palmitic and stearic acids together with a slight decrease in oleic and linoleic acids, no other changes were found in the fatty acid composition of the erythrocyte phospholipids. Anisotropy measurements with n-(9-anthroyloxy) stearic and -palmitic acid fluorophores revealed increased motional freedom of the fatty acid chains in the external lipid layers of the intact erythrocytes from all members of the TPI-deficient family as compared with normal age-matched controls. This asymmetric increase in membrane fluidity was found to be significantly higher in the propositus than in his compound-heterozygote brother without any neurological disorders. The change in membrane fluidity may result from as-yet-unresolved aspects of the lipid composition of the plasma membrane. Our findings that the differences between the TPI-deficient individuals and normal controls and the differences between the two compound-heterozygote brothers were all absent in the phospholipid extracts of the same erythrocytes favor the assumption that the increased motional freedom of the fatty acid chains in the external surface of the bilayer is caused by the binding of the mutant TPI molecule to the N-terminal sequence of band 3 protein.

Hereditary triosephosphate isomerase (TPI) deficiency: two severely affected brothers one with and one without neurological symptoms.

A 13-year-old Hungarian boy (B.J.Jr.) with congenital haemolytic anaemia (CHA) and hyperkinetic torsion dyskinesia was found to have severe triose-phosphate isomerase (TPI) deficiency. One of his two brothers (A.J.), a 23-year-old amateur wrestler, has CHA as well, but no neurological symptoms. Both have less than 10% TPI activity and a highly increased dihydroxyacetone phosphate (DHAP) level in their red blood cells. Their TPI had a slow electrophoretic mobility and was heat unstable. Both parents and a third brother are healthy heterozygous carriers of the defect. A.J. represents a unique phenotype from the point of view that all published "homozygotes" had severe neurological alterations from infancy or early childhood except one infant who died at 11 months, probably too young for neurological symptoms to be noted. In contrast to the two affected Hungarian brothers all but one "homozygote" has died before the age of 6 years. The striking difference in the clinical course of the defect between the two brothers with the same severe red blood cell enzyme deficiency may originate from unusual differences between two double heterozygous brothers resulting inter alia in different levels of TPI expression in various tissues. Significantly lower TPI activities were found in both the T- and B-cells of the propositus as compared to the respective cells of the neurologically symptom-free brother.

[Granulocyte-macrophage progenitor cells in allogenic bone marrow transplantation: correlation of progenitor cell content and regeneration in the graft]. / Granulocyta-makrophag össejtszint vizsgálata allogén csontvelöátültetésben: az oltvány össejttartalma és a regeneráció összefüggése.

The amount of granuloid macrophage progenitors (CFU-GM) was studied in 16 donor bone marrows used for allogenic bone marrow transplantation in the National Institute of Haematology and Blood Transfusion between January, 1984 and January, 1988. In 10 bone marrow transplanted patients long-term follow up of bone marrow CFU-GM regeneration was carried out. Graft sizes were the following: 2.91 +/- 0.62 X 10(8)/kg body weight nucleated cells and 19.2 +/- 14 X 10(4)/kg body weight (CFU-GM. Preinfusion procedures (centrifugation and resuspension) did not alter CFU-GM content of the grafts. Separation of nucleated cells with hydroxyethylstarch, applied for ABO mismatched donor bone marrow, however, resulted in a 30 per cent loss in CFU-GM. Since higher than threshold graft-sizes for successful engraftment were used, no linear correlation between graft size and speed of granulocyte and platelet recovery was found. Significant difference between regeneration kinetics of bone marrow CFU-GM of patients transplanted for CML or AML and ALL was observed: in AML and ALL patients normal bone marrow CFU-GM level was found 4 to 6 months after transplantation, while in CML patients CFU-GM level approached the lower limit of the normal value only 10 to 14 months after transplantation. Granulocyte and thrombocyte recovery of CML patients showed a significant delay when compared to transplanted AML and ALL patients.

Cercopithecus aethiops monkey as a reliable model for in vitro study of T cell depletion of bone marrow with Campath-1 plus complement.

T-lymphocyte markers of peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) of C. aethiops monkeys were studied by using anti-human monoclonal antibodies. The results show that C. aethiops T lymphocytes express surface markers which react specifically with anti-human MoAbs including CD3, CD4, CD8, CD2. However, very few CD3-positive cells were found, in contrast to the abundance in CD8+ cells. There is a high conservation of receptors forming E rosettes with AET-treated SRBCs, and antigens reacting with the anti-human T and B cell monoclonal antibody (Campath-1). The present findings indicate that C. aethiops can be used as a new experimental model for studies on T-cell depletion from bone marrow with Campath-1 MoAb + rabbit C.

Comparison of efficiency of complements from various species for T-cell depletion from Cercopithecus aethiops bone marrow with Campath-1 MoAb in vitro.

Mature T-cells were removed from Cercopithecus aethiops monkey bone marrow with Campath-1 MoAb plus complement from various species (man, rabbit and monkey). The T-cell depletion was more effective and stable with rabbit or human complement than with autologous (monkey) complement. The most complete and effective lytic function to T-cells was found in the case of rabbit complement. Rabbit complement can be used successfully to deplete bone marrow T-cells of C. aethiops with Campath-1 in vitro.

T-cell depletion of Cercopithecus aethiops monkey bone marrow with Campath-1 monoclonal antibody and complement.

The effectiveness of anti-human monoclonal antibody (Campath 1) plus complement (C') in removal of T-cells from Cercopithecus aethiops monkey bone marrow was studied. Recovery of haemopoietic progenitor cells (CFU-GM) was also investigated in vitro after treatment with Campath-1 plus C'. The results showed that the cell-yield was 37.2 +/- 9.8 after Ficoll separation and 44.2 +/- 13.2% after Campath-1 + + C' treatment. The CFU-GM yields referred to the original total CFU-GM were 88.9 +/- 24.0 and 40.0 +/- 14.3%, respectively. After Campath-1 treatment, CFU-GM per 10(5) bone marrow cells was 269.3 as compared to the pretreatment value of 213.0. In the T-cell-depleted bone marrow suspensions mature T lymphocytes could not be detected. Following cryopreservation more than 70% of CFU-GM could be recovered in T-cell depleted bone marrow suspensions kept in frozen state for two months. Cercopithecus aethiops monkeys can be used as a model to study T-cell depletion of bone marrow with Campath-1 plus C' for studying allogeneic bone marrow transplantation.

Gangliosides in human chronic lymphocytic leukaemia.

Gangliosides were analysed by elaborated overpressured thin-layer chromatography (OPTLC) in 6 cell samples from 5 patients with chronic lymphocytic leukaemia (CLL) of B-cell origin, and 4 individual lymphocyte preparations from normal blood donors. The principal difference in the ganglioside profile between these two counterparts appears to be the presence of GD3 and the predominance of the less polar compounds (GM3, GM2, GM1) in CLL cells. Qualitatively, GD3 accounted for about 5.5% of the total CLL gangliosides, whereas it was not detectable in normal lymphocytes. Quantitatively, GM3 constituted more than 81% of the total CLL gangliosides, a proportion more than twice as high as that found in normal lymphocytes. Three other minor gangliosides were isolated from CLL cells; these were shown to be GM2 (trace amounts), GM1 (7.7%), and GD1 (4%). The expression of individual gangliosides varied greatly among the various CLL samples obtained from different patients, and even from the same patient, if examined at different times. No gangliosides were found in the supernatant collected from CLL cells subjected to a temperature shift (0 degrees C to 37 degrees C) or in the cell-free medium harvested from the CLL cells kept in overnight culture.

Logistics of platelet production: platelet concentrate from one-day-old blood stored at 10 degrees C.

Platelet concentrates were prepared from blood stored at 18 to 20 hours at either 10 +/- 2 degrees C or 16 +/- 2 degrees C. Platelets prepared from blood stored at 10 degrees C had good in vitro properties. The results were not distinguishable from those obtained with freshly-isolated platelets. The in vivo effectiveness of platelets prepared in this manner must be established.

Plasmapheresis in patients with leukaemia, multiple myeloma and immune complex diseases.

Therapeutical partial plasma exchange was performed on 9 patients with acute leukaemia, 14 patients with monoclonal and polyclonal gammopathies and 3 with primarily immune complex disease. The procedure was effective in 21 out of the 27 courses performed on patients with gammopathies and in all cases of the primarily immune complex diseases. In acute leukaemic patients the course of the disease was favourably influenced by plasmapheresis. The results show that partial plasma exchange is an effective adjunct therapy in the treatment of patients with haematological diseases.

Platelet phospholipids in thrombocytosis due to myeloproliferative disorders.

Platelet phospholipids were analyzed in patients with thrombocytosis due to myeloproliferative disorders and secondary thrombocytosis. A significant increase of phosphatidylcholine together with a decrease of sphingomyelin was observed in each of the 8 patients with primary thrombocythaemia and in each of the 5 patients with excessive thrombocytosis due to primary polycythaemia. The phospholipid pattern of the 3 patients with secondary thrombocytosis did not differ from that of the normal controls. This marked difference in the platelet phospholipids may be helpful in distinguishing secondary thrombocytosis persisting for a prolonged period of time after splenectomy from previously unrecognized primary thrombocythaemia unmasked by splenectomy. The decreased sphingomyelin/lecithin ratio of platelets in primary thrombocythaemia may interfere with platelet function by increasing the fluidity of their membrane.

The effect of Na3-citrate on the solubility of cryoprecipitate (citrate effect on cryoprecipitate).

Cryoprecipitate stored frozen could be redissolved only if it contained more than 10 mmol/l Na3-citrate. It is shown that in the insoluble residue less than 10 mmol/l Na3-citrate is fibrinogen. In saline containing 10 to 20 mmol/l Na3-citrate after thawing, the cryoprecipitate was dissolved entirely.

Control of life-threatening bleeding by combined plasmapheresis and immunosuppressive treatment in a haemophiliac with inhibitors.

Life-threatening lingual haemorrhage was successfully treated in a 3-year-old haemophiliac who had a high level of inhibitor in his plasma. Repeated plasma exchange with high-dose factor VIII concentrate led to a cessation of bleeding. Immunosuppressive treatment introduced at the time of plasmapheresis and carried out for 6 weeks seems to have prevented the return of the inhibitor.

The effect of increasing glucose in CPD on the quality of stored blood.

Changes in quality of blood units containing one and a half or double amounts of glucose, stored at +4 degrees C for three weeks were analysed. An experimental preservative containing glucose and fructose (1 : 1) was also used. No other additives (purine or purine-nucleoside) were applied. A standard CPD preservative of the National Inst. of Haematology and Blood Transfusion was used as control. The pH, plasma free haemoglobin, K+ content, red blood cell (RBC) ATP and 2,3-DPG content, and RBC fragility index were determined in each sample. Increase of glucose concentration, the addition of fructose had a beneficial effect on blood pH, and on plasma free haemoglobin and K+ concentration. 150% glucose improved the 2,3-DPG maintenance in stored blood.