Individual antigenic specificity of human macroglobulins: Localization of antigenic determinants and their use to demonstrate formation of hybrid molecules after reduction and reassociation.

The localization of individually specific antigenic determinants on the monoclonal gammaM Mö was studied in gel diffusion experiments with 19S gammaM Mö and its 7S subunit. Various types of such determinants were delineated. Some were demonstrable both on the intact macroglobulin molecule and on the subunit. The latter was often unable to precipitate with the corresponding antibody, but combined with it and inhibited precipitation. Immunization of rabbits with purified gammaMs Mö was the most useful way to obtain antisera demonstrating individually specific antigenic determinants on the subunit by direct precipitation. Still other determinants were apparently destroyed by mild reduction of the gammaM molecule. Reduction and reassociation of mixtures of two monoclonal gammaM-globulins resulted in formation of hybrid molecules containing subunits of different origin. Individually specific antisera against the two components were useful for demonstrating hybrid formation. Serological activity, i.e., cold agglutinin activity, was present in the hybrid molecules.

Inhibition of the Fc receptor of human lymphoid cells by antisera-recognizing determinants of the hla system.

The inhibitory effect of HLA antisera on Fc receptors of human lymphoid cells was investigated. The ability of lymphoid cells to form rosettes (FcRFC) with antibody-coated sheep red blood cells and to function as effector cells (K cells) in antibody-dependent cell-mediated cytotoxicity were used as assay systems. We found that antisera recognizing determinants of the HLA-A, -B, and -C series had no effect on FcRFC, while a specific inhibitory effect was observed of antisera probably reacting with determinants identical to or closely associated with those of the HLA-D series. This inhibitory effect was retained in the F(ab')2 fragments. Specific inhibition of K cells was observed with all HLA antisera, but this effect was lost in the F(ab')2 fragments. We conclude that the Fc receptor of rosette-forming lymphocytes may be closely associated with products of the HLA-D region. This is analogous to the association between the Fc receptor and the Ia antigens on murine rosette-forming B lymphocytes.

Ultradian rhythms in Arabidopsis thaliana leaves in microgravity.

Ultradian movements of Arabidopsis thaliana rosette leaves were discovered and studied under microgravity conditions in space. Weightlessness revealed new facets of these movements. The European Modular Cultivation System (EMCS) was used in a long-term white-light, light-darkness (LD; 16 : 8 h) experiment on the International Space Station (ISS). Leaves reacted with slow up or down movement (time constant several hours) after transitions to darkness or light, respectively. Superimposed movements with periods of c. 80-90 min and small-amplitude pulsed movements of 45 min were present in the light. Signal analysis (fast Fourier transform (FFT) analysis) revealed several types and frequencies of movements. Identical phase coupling was observed between the 45-min movements of the leaves of one plant. In darkness, movements of c. 120-min period were recorded. The EMCS allowed 0-g to 1-g transitions to be created. Leaves on plants germinated in microgravity started a negative gravitropic reaction after a delay of c. 30 min. Leaves grown on a 1-g centrifuge reacted to the same transition with an equal delay but had a weaker gravitropic response. The experiments provide unequivocal demonstrations of ultradian, self-sustained rhythmic movements in A. thaliana rosette leaves in the absence of the effect of gravity.

Gravity amplifies and microgravity decreases circumnutations in Arabidopsis thaliana stems: results from a space experiment.

In a microgravity experiment onboard the International Space Station, circumnutations of Arabidopsis thaliana were studied. Plants were cultivated on rotors under a light:dark (LD) cycle of 16 : 8 h, and it was possible to apply controlled centrifugation pulses. Time-lapse images of inflorescence stems (primary, primary axillary and lateral inflorescences) documented the effect of microgravity on the circumnutations. Self-sustained circumnutations of side stems were present in microgravity but amplitudes were mostly very small. In darkness, centrifugation at 0.8 g increased the amplitude by a factor of five to ten. The period at 0.8 g was c. 85 min, in microgravity roughly of the same magnitude. In white light the period decreased to c. 60 min at 0.8 g (microgravity value not measurable). Three-dimensional data showed that under 0.8 g side stems rotated in both clockwise and counter-clockwise directions. Circumnutation data for the main stem in light showed a doubling of the amplitude and a longer period at 0.8 g than in microgravity (c. 80 vs 60 min). For the first time, the importance of gravity in amplifying minute oscillatory movements in microgravity into high-amplitude circumnutations was unequivocally demonstrated. The importance of these findings for the modelling of gravity effects on self-sustained oscillatory movements is discussed.

Update on pathogen reduction technology for therapeutic plasma: an overview.

Human plasma for therapeutic use, besides having optimal viral safety, must contain optimal levels of all coagulation factors and protease inhibitors to be clinically effective. Several new technologies for pathogen reduction of plasma (PRT) exist and are entering the stage of clinical testing. The main objective of this overview is to provide an update on the current states of three promising photoactive technologies that target pathogen nucleic acid for pathogen inactivation, applicable to single unit fresh-frozen plasma (FFP) and to highlight the experiences gained with classical pathogen reduction of pooled plasma using solvent-detergent (SD) treatment. It should be emphasized that none of the currently applied methods inactivate all types of pathogens and all have some effect on plasma quality when compared to fresh-frozen plasma. Pooled SD-plasma is the best documented clinical product, followed by methylene blue light treated (MBLT)-plasma. Recently, Psoralen light treated (PLT)-plasma has been introduced (CE-marked product in Europe) while Riboflavin light treated (RLT)-plasma is still under development. In principal, PRT for plasma not only differs in terms of the spectrum and log of pathogen reduction potential, but also in respect to the physicochemical/biological characteristics, and profiles of the adverse reactions, particularly in vulnerable patient groups. Therefore, an additional practical step such as oil extraction followed by chromatography to remove the solvent/detergent, and filtration or the use of some special absorbing matrix is required to reduce the residual photosensitive chemicals, their metabolites and photo adducts. This is required to improve the safety margin of the final product. Moreover, while it may be convenient to think that a combined pathogen reduction technology could improve the spectrum of known pathogens to be inactivated, one needs, in practice, to balance between the degree of pathogen reduction and the loss of some plasma protein activity. From the quality point of view, SD-plasma is a pooled standardized pharmaceutical product with extensive in-process control. However, both differences in production processes and the plasma source can influence final product quality. On the other hand, single unit plasma derived from nucleic acid PRT cannot be monitored by pharmaceutical process control and demonstrates the wide range of concentrations normally observed for plasma proteins. Pooling has the disadvantage that one single plasma unit can contaminate a whole pool, but this can be offset by several advantages that pooling and the SD process offer. Among these are reduction of a possible pathogen load by dilution and by neutralizing antibodies in the plasma pool, dilution and possible neutralization of antibodies and allergens which essentially eliminates transfusion-related acute lung injury (TRALI) and reduces allergic reactions significantly, removal of residual blood cells, cell fragments and bacteria, and removal of the largest von Willebrand-factor (vWF) molecules. On the other hand, some streamlining is required for technologies using single units of plasma, such as the use of plasma from male non-transfused donors to reduce TRALI and to avoid the O blood group in order to meet current specifications for FFP [Seghatchian J. What is happening? Are the current acceptance criteria for therapeutic plasma adequate? Transfus Apheresis Sci 2004; 31:67-79], and to exploit the potential benefit to inactivate residual lymphocytes and prevent transfusion-associated graft versus host disease. The cost effectiveness of pathogen inactivation is very low (> 2 million US dollar/life year saved), if however, non-infectious complications such as TRALI are taken into account; the cost for SDP is reduced to < 50,000 British pound/life year saved for those 48 years. Finally, from the therapeutic standpoint, two important questions still remain to be answered. First, whether the various pathogen reduced plasma products are clinically interchangeable and second, whether the conventional quality requirements of FFP are still adequate for the newer plasma products. These questions can only be answered by a head to head comparison, followed by large-scale clinical trials.

Rational use of blood products.

Blood product transfusions can be life saving and must be considered in the supportive care of children of any age with underlying oncological or haematological problems, as well as after major surgery or after serious trauma. Paediatric transfusions are particularly challenging because life-long effects of transfusion complications are more durable and serious in children than in adults, in whom the median age at transfusion is >70 years (Tynell E, Norda R, Shanwell A, Björkman A. Long-term survival in transfusion recipients in Sweden, 1993. Transfusion 2001, 41, 251-255). While the general indications for transfusions in paediatric patients are similar to adults, the threshold, volumes and infusion rates for transfusions vary with age. In this Update, we discuss current blood products, then suggest transfusion "triggers" in major surgery and haematological and oncologic practice. Finally, future developments and new possibilities are considered.

Association of beta-2-microglobulin and HL-A in the cell membrane. A general property in human cells?

To study whether the recently reported close association of beta-2-microglobulin and HL-A determinants is restricted to lymphocyte membranes or might be a more general property, experiments were performed where normal and malignant human cells were tested for coaggregation-capping of the two determinants. The normal cells were fibroblasts and the epithelial cells were from skin. The turmor cell lines were HeLa clone S3 carcinoma cells and National Collection of Type Cultures strain 3075, which is a line of malignant human skin cells. The cells were double-stained with fluorescein-labelled conjugates to demonstrate the reaction of anti-HL-A and anti-beta-2-microglobulin with the respective cell membrane components. Aggregation and capping of the HL-A and beta-2-microglobulin determinants in the cell membrane were always observed after incubation at 37 C for 30 min. The red and green fluorescence were always superimposed when anti-beta-2-microglobulin was used in the first (aggregating) incubation step. This demonstrates that the HL-A and beta-2-microglobulin determinants are also associated in the cell membrane of cells other than lymphocytes.

Effect of blood transfusions on renal transplantation: study of 191 consecutive first transplants from living related donors.

One hundred ninety-one consecutive living related transplants performed from 1969 to the end of 1978 have been analyzed for the effect of pretransplant blood transfusions. Superior graft survival was observed in transfused patients transplanted with a one HLA haplotype-disparate kidney, whereas no effect of blood transfusions could be observed on the survival of HLA-identical transplants. The frequency of first rejection episodes was significantly reduced in transfused compared to nontransfused one haplotype-mismatched transplants, while no influence of blood transfusions was seen in patients with HLA-identical transplants. The survival of patients was, however, not influenced by previous transfusions. Pretransplant hemodialysis improved graft survival and patient survival; the difference was, however, only significant at 2 years in the one haplotype-mismatched group. When analyzed separately, both blood transfusions and hemodialysis had a beneficial effect on graft survival in one haplotype-mismatched transplants.

Significance of HLA matching in renal transplantation. A prospective one-center study of 485 transplants matched or mismatched for HLA-A, B, C, D, and DR antigens.

Matching for HLA haplotypes as well as for HLA-A and B antigens improved graft survival in 112 living related first transplants. In cadaveric first transplants, matching for HLA-A and B antigens had a beneficial effect on the fate of 373 grafts, while matching for HLA-C antigens had no predictive value. One hundred seventeen cadaveric transplants and their recipients were prospectively typed for the HLA-DR antigens. Compatibility for HLA-DR was found to be prognostically beneficial irrespective of matching for HLA-A and B antigens, and with no difference between transfused and nontransfused patients. Matching both for HLA-A , B and D/DR was thus found to influence the outcome of renal transplantation.

Detection of HLA-D region products by primed lymphocyte typing (PLT).

In vitro priming experiments were performed with lymphocytes from members of two different families carrying Dw-,DR2 or Dw2,DR2 haplotypes. It was demonstrated that lymphocytes could be primed to allogeneic HLA-D determinants without detectable priming to the associated HLA-DR determinant, even when the priming cell was also HLA-DR incompatible to the responding cell. It was further shown that the unknown HLA-D determinants of the two families (e.g., Dw-) were different, one of them showing cross-reactivity to Dw2. Priming to MT1 determinants or to Lewis antigens could not be detected.

A randomised study of allogeneic transplantation with stem cells from blood or bone marrow.

Sixty-one consecutive adult patients with leukaemia, primary myelofibrosis or myelodysplastic syndrome with an HLA-identical or one antigen mismatched family donor were randomised to allogeneic transplantation with PBPC or BM. Progenitor cells were mobilised into the blood by giving the donors 10 microg/kg/day G-CSF subcutaneously for 5-7 days. G-CSF was not given to patients after transplantation. The time to neutrophil counts >0.5 x 109/l was 17 days (95% CI 15.2-18.8 days) in the PBPC group compared to 23 (95% CI 20.3-25.7 days) in the BM group (P = 0.0005). The time to platelet counts >20 x 109/l was 13 days (95% CI 11.7-14.3 days) in the PBPC group and 21 days (95% CI 18.7-23.3 days) in the BM group (P = 0.0005). Acute GVHD of grades II-IV developed in six patients transplanted with PBPC and three patients transplanted with BM. The numbers of patients with chronic GVHD were 15 and 8, respectively. Transplant-related mortality and leukaemia-free survival showed no significant differences. Transplantation with PBPC appears preferable for the recipient due to faster neutrophil and platelet recovery. However, the final conclusion can not be drawn before long-term results on chronic GVHD and relapse incidence in longer randomised trials are available.

Donation of stem cells from blood or bone marrow: results of a randomised study of safety and complaints.

Biological consequences and physical complaints were compared for donors randomly assigned either to blood stem cell (BSC) or bone marrow (BM) donation. In the period 1994-1999, 61 consecutive donors were included. The BSC donors were given G-CSF 10 microg/kg s.c., daily during 5 days before the first leukapheresis. Nineteen donors had one leukapheresis, 10 required two and one donor needed three leukaphereses in order to reach the target cell number of 2 x 10(6) CD34(+) cells/kg bw of the recipient. A median platelet nadir of 102 x 10(9)/l was reached shortly after the last leukapheresis. Three weeks post harvest, 17 of 30 BSC donors had a mild leukopenia. Six had a leukopenia lasting more than a year before returning to normal values. Both groups were monitored prospectively through a standardised questionnaire completed by the donors. BSC donation was significantly less burdensome than BM donation and was preferred by the donors. The short-term risks of BSC mobilisation and harvest seem negligible. The potential long-term effects of G-CSF are unresolved and the donors must be followed closely.

Fewer relapses and increased chronic GVHD in patients transplanted with blood stem cells: a 5-year follow-up in a single centre study.

A total of 61 consecutive adult patients with haematological malignancies with an HLA-identical or one antigen-mismatched haploidentical family donor were randomised to allogeneic transplantation with blood stem cells (BSC) or bone marrow (BM). The median observation time was 5 years. Apart from engraftment parameters and acute graft-versus-host disease (GVHD), transplant-related mortality (TRM), incidence and severity of chronic GVHD, relapse, leukaemia-free survival (LFS) and overall survival (OS) were recorded. In the BSC and BM group, respectively, TRM was 8/30 and 4/30 (P=0.405), the incidence of chronic GVHD was 15/26 and 11/30 (P=0.138), extensive chronic GVHD was 10/26 and 4/30 (P=0.034), and relapse one and 10 patients (P=0.007). In log-rank test restricted to the cases allografted from HLA-identical donors, the difference remained significant with regard to relapse incidence (P=0.039), but not extensive chronic GVHD (P=0.072). No difference in LFS and OS was observed. In conclusion, our study strongly indicates an enhanced graft-versus-leukaemia effect in BSC recipients, which is not expressed in increased survival. The increased chronic GVHD in these patients may contribute, but the relation is complex and not yet understood.

HLA antigens in multiple sclerosis.

The frequency of the MLC activating HLA-Dw2 determinant was found significantly increased to 68% among 37 patients with long-standing multiple sclerosis, compared to 30.2% among healthy controls. A similar (or stronger) degree of association was found with an "Ia-like" B cell specificity, SOW, while the association with HLA-B7 was less apparent. No correlation between HLA-Dw2 and signs of local synthesis of oligoclonal IgG or measles or rubella virus antibodies in the CNS was observed.

The Norwegian Plasma Fractionation Project--a 12 year clinical and economic success story.

The establishment of the Norwegian Fractionation Project (Project) was of major importance in preserving national self-sufficiency when plasma, cryoprecipitate and small batch factor IX-concentrates were replaced by virus inactivated products in the last part of the 1980s. Fractionation was performed abroad by contract with Octapharma after tenders on the European market. All Norwegian blood banks (>50) participated in the Project. Total yearly production was 50-60 tons of mainly recovered plasma. From 1993 solvent detergent (SD) treated plasma has replaced other plasma for transfusion. The blood banks paid for the fractionation and/or viral inactivation process, while the plasma remained the property of the blood banks and the final products were returned to the blood banks. The Project sold surplus products to other Norwegian blood banks and the majority of the coagulation factor concentrates to The Institute of Haemophilia and Rikshospitalet University Hospital. Both plasma and blood bank quality was improved by the Project. Clinical experience with the products has been satisfactory and self-sufficiency has been achieved for all major plasma proteins and SD plasma, but a surplus exceeding 3 years consumption of albumin has accumulated due to decreasing clinical use.The Project has secured high yields of the fractionated products and the net income from the produced products is NOK 1115 (140 Euros or US dollars) per litre plasma. An increasing surplus of albumin and the possibility of significant sales abroad of currently not fractionated IVIgG, could lead to a reorganisation of the Project from that of a co-ordinator to a national plasma handling unit. This unit could buy the plasma from the blood banks and have the plasma fractionated by contract after tender, before selling the products back for cost recovery. The small blood banks could produce plasma for products for the Norwegian market, while surplus products from the larger blood banks which are certified for delivery of plasma for fractionation of products to be consumed in the European Community, could be sold on the international market.

Clinical implications of red blood cell and platelet storage lesions: an overview.

Both red blood cells and platelets undergo lesions upon storage which affect their function and possibly their clinical outcome. Some of these lesions are reversible, others not. Improved additive solutions and leukocyte depletion can delay the appearance of storage lesions. In addition, cellular apoptosis leads to numerous mitochondrial and surface changes during storage which have the potential to induce immune suppression by tuning down the innate immune system. This overview highlights some laboratory and clinical aspects of red cell and platelet storage lesions.

Improved preservation of coagulation factors after pre-storage leukocyte depletion of whole blood.

Plasma and red blood cell quality are affected both by citrate concentration and the levels of extracellular leukocyte and platelet derived substances, accumulated during storage of blood. The effect of leukocyte filtration on the storage stability of whole blood was therefore studied in blood collected in standard CPD and 0.5CPD (CPD with half strength citrate concentration). A total of 52 units, 12 of them with reduced citrate concentration, were leukocyte-filtered with Pall( whole blood filter (WBF1 or 3). No differences in leukocyte or platelet reduction were observed with the two citrate concentrations. However, with 0.5CPD a significantly longer filtration time and increased complement activation was observed. The effect of pre-storage leukocyte filtration on the plasma quality of whole blood was therefore only studied with standard CPDA1 anticoagulant solution (normal strength citrate concentration). Leukocyte filtration did not affect the von Willebrand factor concentration, while a small reduction (7%, p=0.04) in factor VIII (FVIII) concentration was observed. During storage, however, FVIII decreased more slowly in the filtered than in the unfiltered product, and, from day two, the FVIII content was significantly higher in the filtered product (46% versus 30% at 28 days, p<0.001). Factor V (FV) demonstrated a 16% reduction (p<0.001) upon filtration, followed by an additional 8% in the next 24 h and only a 4% reduction the next 27 days, while unfiltered products demonstrated a continuous reduction to 26% at 28 days. While the beta-thromboglobulin (beta-TG) concentration significantly increased (from 836 to 2483 IU/ml, p<0.001) during leukocyte filtration, no further increase was observed during storage. In contrast, unfiltered products demonstrated an increase to 5762 IU/ml (p<0.001) at 14 days, followed by a slight, not significant, reduction. This indicates platelet activation during filtration and explains a parallel reduction in FV. Filtration induced no increase in prothrombin fragment 1+2, while a slight increase was observed in some unfiltered products after 28 days of storage.Pre-storage leukocyte depletion thus improves the coagulation factor content of plasma in stored whole blood.

Transferrin receptor in serum. A new tool in the diagnosis and prevention of iron deficiency in blood donors.

BACKGROUND: Transferrin receptor mediates cellular uptake of iron, and the expression on cells reflects iron needs and erythropoietic activity. The results of measuring transferrin receptor in serum (sTfR) in blood donors are presented. STUDY DESIGN AND METHODS: Haemoglobin, serum-ferritin and sTfR were measured in 172 female and 174 male donors that had donated whole blood six or more times during the previous 3 years and in 96 female and 56 male new donors. RESULTS: Haemoglobin and sTfR were not significant different in new and repeat donors. New donors had significantly higher s-ferritin than repeat donors. Twenty donors had a Hb above the low limit for normal, but below the determined cut-off for donation. Only three of these had high sTfR and/or low serum-ferritin. Hence, of the total 492 donors 3.5% were below the Hb cut-off, but having Hb, s-ferritin and sTfR within normal ranges. 11.6% of new female donors belonged in this category. CONCLUSION: STfR is better than s-ferritin as a screening for iron deficiency. Most donors with low tissue iron neither have high sTfR, nor anaemia. There is probably no need to have a separate, higher than the lower normal range, requirement for Hb in donors. STfR measurements are probably most valuable in a setting where most donors are repeat donors.

Close association between Fc receptor and HLA-D-associated (Ia-like) determinants on human lymphoid cells.

The inhibitory effect of HLA antisera on Fc receptors of human lymphoid FcRFC and K cells was investigated. Antisera recognizing determinants of the HLA-A, -B, and -C series had no effect on FcRFC, while specific inhibition was observed with an antiserum reacting with determinants closely associated to HLA-DW2. This inhibitory effect was also demonstrated by the Fab' fragments. Specific inhibition of K cells was observed with all HLA antisera, but this effect was lost in the Fab' fragments. We concluded that the Fc receptor of FcRFC may be closely associated with products of the HLA-D region. This is analogous to the association between the Fc receptor and the Ia antigens on murine splenic B lymphocytes.