IgE-mediated anaphylactic reaction to purified and recombinant factor VIII in a patient with severe haemophilia A.

We report a 51-year-old patient with severe haemophilia A developing a severe life-threatening anaphylactic reaction to recombinant factor VIII (rFVIII). Anaphylactic reactions are a rare but well-known side effect of FVIII products. The nature of these reactions could not be clarified as previous studies failed to demonstrate a specific IgE response. Here, we could prove a grade 3 anaphlyactic reaction as an IgE-mediated response to rFVIII for the first time by Western blotting.

[Quality of washed autologous erythrocytes from drainage-suction pumps]. / Qualität gewaschener autologer Erythrozyten bei Einsatz von Drainage-Saugpumpen.

High sub-pressure in high-vacuum suction bottles falls as the bottles fill up. Suction pumps with reservoir have a constant low suction level and decisive advantages. The question is: does the use of a suction pump before processing and retransfusion influence the quality of the erythrocytes? The randomized, controlled, prospective study presented here deals with drainage blood and washed autologous red blood cells (warbc) from 60 patients after hip endoprosthesis surgery. In a comparison between suction pump and redon bottle, the following parameters were studied: haematological-parameter (haemoglobin, haematocrit, erythrocyte count, leukocyte count, thrombocyte count, MCV, MCH, MCHC), vitality (osmotic fragility, 2,3-DPG) and haemolysis parameter (GOT, LDH, plasma haemoglobin, potassium). Control samples were taken immediately after operation: sample one from drainage blood before processing and sample two from warbc before retransfusion. There were no significant statistical differences between the groups. The osmotic fragility of the retransfused red blood cells was slightly above normal values, while the 2,3-DPG was normal. "Old" erythrocytes were haemolysed. The concentration of plasma haemoglobin was clearly above the normal range. In the "redon group" GOT and LDH were clearly increased. The quality of erythrocytes from suction pump reservoirs is not decisively impaired.

Leukocyte-reduced blood products.

During the past 10 years, methods for blood component preparation have undergone considerable change. Refinements of traditional methodologies were necessary in response to demands for increasingly effective and safe platelet support for the intensive chemotherapy for patients with malignant disease, as well as a response to evidence of the immunological effects of whole blood or unmanipulated erythrocytes. At the center of the issue is concern for the consequences of transfusing contaminant leukocytes. Clearly, for all transfusions the lowest possible leukocyte content is desirable, and their removal should be as early as possible during storage. This review of recent journal articles focuses on reports of investigations of the effects of allogeneic leukocytes in blood transfusions. There is a sense of competition among the advocates for secondary elimination of leukocytes (by filtration) versus those favoring the approach of primary avoidance of leukocyte contamination (by centrifugation). As the following summaries indicate, the relative merits of leukocyte reduction by filtration, either before or after storage, versus leukocyte reduction by specialized apheresis methods remain undefined. The goal of a universal guideline for the use of leukocyte-reduced blood components is not yet achieved.

Therapeutic immunoadsorption--its role in clinical practice.

Extracorporeal immunoadsorption onto staphylococcal protein A replaces conventional plasmapheresis because its elimination efficiency is higher while less adverse reactions are seen. Experience in immunoadsorption is limited to specialized centers although recent technical developments make the procedure simple and safe to use. During a workshop held in Munich, September 1996, the application of immunoadsorption in various diseases was discussed. Goodpasture's syndrome, acquired autoimmune coagulopathy, HLA-sensitized transplantation and treatment-resistant life-threatening autoimmune diseases were accepted indications for immunoadsorption. Immunoadsorption in other immunoglobuline and immune complex mediated diseases is still under discussion.

Immunological effects of therapeutic immunoadsorption with respect to biocompatibility.

The activation of the complement system leading to generation of anaphylatoxins and the membrane attack complex depends on the chemical nature of the adsorptive system and the anticoagulation used. The method of the primary separation determines the presence of cell debris in the plasma as well as the extent of platelet activation. The particular role of anticoagulation and its properties to prevent/reduce complement activation on immunadsorption material is discussed and the combined use of citrate and heparin is proposed. The quality of the reinfused plasma--as discussed on the example of LDL-apheresis--is therefore influenced by the amount of the activated split products. This determines finally the extent of cellular activation during therapeutic immunadsorption when receptor-dependent activation of cells by C3a(desarg) and C5a(desarg) can occur.

Imprecision of counting CFU-GM colonies and CD34-expressing cells.

Determinations of committed haemopoietic progenitor cells, namely CFU-GM (colony-forming unit-granulocyte/macrophage) and of CD34-expression haemopoietic cells as assessed by multiparameter flow cytometry are routine diagnostic tools in haemopoietic cell therapy. Generally, the tests are used to optimise the timing and management of cytapheresis and to assess the engraftment potential of the harvested cells. Both measurements, however, are at best surrogate markers, as an adequate routine test which effectively assesses the short- and long-term repopulating haemopoietic cell is not available. Nonetheless, cell threshold doses have been established. Above these thresholds rapid engraftment is almost invariable but below these thresholds the outcome is variable. In this study we have focussed on the imprecision in counting haemopoietic cells, as assessed as CFU-GM and as CD34-expressing cells. The data on both tests have been analysed from six European institutions. The coefficient of variation in CFU-GM colony counting was about 30%, whereas the coefficient of variation in flow cytometric counting of CD34-expressing cells was about 10%. These data suggest that the technical imprecision in enumerating progenitor cells, particularly CFU-GM, at low levels, might make a major contribution to the clinical variability observed after transplantation of sub-threshold progenitor cell dose.

Simultaneous genotyping of human platelet antigens by hot start sequence-specific polymerase chain reaction with DNA polymerase AmpliTaq Gold.

OBJECTIVES: Human platelet alloantigen (HPA) typing has potential clinical relevance in a variety of contexts. We can improve methods for HPA genotyping by complementing our knowledge of the DNA sequence polymorphisms of HPA genes and experience with various DNA-based HPA typing techniques. METHODS: A newly available DNA polymerase, AmpliTaq Gold (Perkin Elmer), provided in an inactive state and activated by heat, makes it possible to perform a hot start polymerase chain reaction (PCR) in order to prevent nonspecific amplification during the setup of PCR. To establish a practical procedure for HPA-1, 2, 3 and 5 genotyping, we applied the AmpliTaq Gold for a hot start PCR and employed 8 pairs of published sequence-specific primers (SSP). A simple simultaneous genotyping of these 4 HPA systems could be rapidly achieved with high specificity. RESULTS: The HPA gene frequencies observed in 126 randomly selected German blood donors were 0.82 and 0.18 for HPA-1a and 1b, 0.92 and 0.08 for HPA-2a and 2b, 0.63 and 0.37 for HPA-3a and 3b and 0.90 and 0.10 for HPA-5a and Sb, respectively. CONCLUSION: Using our hot start PCR-SSP procedure with AmpliTaq Gold a simple, rapid and reproducible genotyping for HPA-1, 2, 3 and S systems could be achieved.

Technical and safety aspects of blood and marrow transplantation using G-CSF mobilized family donors.

An allogeneic transplantation programme using immunoselected blood progenitor and bone marrow CD34+ cells has been established. Thirteen healthy HLA-matched, MLC negative sibling donors received two doses of 5 micrograms kg-1 G-CSF (s.c. daily) for 5 days. On days 4 and 5, large-volume mononuclear cell aphereses were performed (COBE Spectra) and on day 5 one unit of autologous blood was obtained. Mononuclear cells were pooled and cryopreserved after CD34+ cell-immunoselection on day 5. Bone marrow (BM) of the same donors was procured under routine conditions 10-45 days later (median: 27 days). The final graft consisted of blood CD34+ cells with either complete BM (n = 5) or immunoselected BM CD34+ cells (n = 8). The present paper describes the progenitor cell mobilization and apheresis protocol and analyzes the cell loss by BM and peripheral blood progenitor cell (PBPC) donation. Considerably larger amounts of mononuclear cells (CD45+), T-lymphocytes (CD3+) and platelets were lost by the apheresis as compared to bone marrow without apparent immediate clinical consequences for the donors. Owing to cross-cellular contamination of the apheresis concentrate, blood platelet count (PC) significantly decreased (mean PC after the second apheresis 116 x 10 microL-1); furthermore on average 3.04 x 10(10) CD3+ cells were removed by two apheresis sessions. This loss did not lead to long-term total lymphocyte count changes (2370 microL-1 versus 1889 microL-1) as observed during the long-term follow-up of 7/13 donors (mean 290 days). Subjectively, the PBPC collections were better accepted than BM donations in all but one family donor.

Transplantation of allogeneic CD34+ blood cells.

Pluripotent stem cells of hematopoiesis and lymphopoiesis are among the CD34+ cells in blood or bone marrow. After granulocyte-colony stimulating factor (G-CSF) treatment, 1% to 2% of the mononuclear cells in blood are CD34+ cells, which can be procured by leukapheresis. We investigated the potential of CD34+ blood cells for reconstituting hematopoiesis and lymphopoiesis after allogeneic transplantation. HLA-identical sibling donors of 10 patients with hematologic malignancies were treated with G-CSF (filgrastim), 5 microgram/kg subcutaneously twice daily for 5 to 7 days. CD34+ cells were selected from the apheresis concentrates by immunoadsorption, concomitantly the number of T cells was reduced 100- to 1,000-fold. After transplantation, five patients received cyclosporine A for graft-versus-host disease (GvHD) prophylaxis (group I); five patients additionally received methotrexate (group II). G-CSF and erythropoietin were given to all patients. Mean numbers of 7.45 x 10(6) CD34+ and 1.2 x 10(6) CD3+ cells per kilogram were transplanted. In group I, the median times of neutrophil recovery to 100, 500, and 1,000 per mm3 were 10, 10, and 11 days, respectively. Group II patients reached these neutrophil levels after 10, 14, and 15 days, respectively. Platelet transfusions were administered for a median of 18 days in group I and 30 days in group II, and red blood cells for 9 and 12 days, respectively. Between day 30 and 60, lymphocytes reached levels of 353 +/- 269 cells per mm3. The median grades of acute GvHD were III in group I and I in group II. Two patients in group I died from acute GvHD. Two leukemic relapses occurred in group II. Complete and stable donor hematopoiesis was shown in all patients with a median follow up of 370 (45 to 481) days. Allogeneic blood CD34+ cells can successfully reconstitute hematopoiesis and lymphopoiesis. Reduction of T cells by CD34+ blood cell enrichment and cyclosporine A alone might not be sufficient for prophylaxis of severe acute GvHD.

[Ex vivo increase in hematopoietic progenitor cells for clinical use]. / Ex-vivo-Vermehrung von hmatopoetischen Progenitorzellen für die klinische Anwendung.

BACKGROUND: The clinical use of ex vivo expanded hematopoietic progenitor cells is currently explored. MATERIAL AND METHODS: In this study the culturing of G-CSF mobilized and purified CD34+ blood cells was investigated. The interleukins IL-1 beta, IL-3 and IL-6 (each at a dose of 300 U/ml) and stem cell factor (25 ng/ml) without or with erythropoietin (1 U/ml) were used. Cells of 10 healthy sibling donors and 10 patients with solid tumors were incubated under small-scale (n = 15, 2 ml) and large-scale (n = 7, 50 ml) culture conditions at 37 degrees C for 5 and 4 weeks, respectively. The cell density was adjusted to about 1 x 10(5) cells/ml. RESULTS: The nucleated cell counts increased approximately 7-fold and 10- to 70-fold after 1 and 2 weeks of incubation. Numbers of CD34+ cells doubled to triplicated within this time interval, without any significant changes in their clonogenicity (CFU-GM and BFU-E output). Thereafter a depletion of the CD34+ cell pool was noticed. However the numbers of CD34+/CD38(-)- or CD34+/ HLA-DR(-)- cells were reduced to a lesser extent. The expanded cells generated predominantly myeloid and almost no lymphoid cells. More glycophorin-A+ cells were produced when erythropoietin was added. Replacement of non-human additives with heat-inactivated autologous plasma had no influence on cell growth. Almost no proliferation was obtained with a 10-fold higher cell density (1.7 x 10(6)/ml in 100 ml), but the cells maintained their viability for 13 to 16 days. CONCLUSIONS: This study suggests, that the chosen culture conditions might be feasible for a large-scale ex vivo expansion of hematopoietic progenitor cells for clinical application. The impact of the ex vivo generated cells on hematopoietic regeneration after chemotherapy is currently under clinical investigation.

Combined transplantation of allogeneic bone marrow and CD34+ blood cells.

Allogeneic peripheral blood progenitor cells (PBPCs) were transplanted after immunoselection of CD34+ cells. Two patient groups were studied: group I patients received immunoselected blood CD34+ cells and unmanipulated marrow cells from the same donor. Group II patients were given immunoselected blood and bone marrow (BM) CD34+ cells. One to 6 weeks before bone marrow transplantation (BMT), PBPCs from HLA-identical and MLC- sibling donors were mobilized with granulocyte colony-stimulating factor (G-CSF) (5 micrograms/kg twice daily subcutaneously) for 5 days. Aphereses were performed at days 4 and 5 of G-CSF application. CD34+ cells were separated from the pooled PBPC concentrates by immunoadsorption onto avidin with the biotinylated anti-CD34 monoclonal antibody 12.8 and then stored in liquid nitrogen. BM was procured on the day of transplantation. Patients were conditioned with either busulfan (16 mg/kg) or total body irradiation (12 Gy) followed by cyclophosphamide (120 mg/kg). Cyclosporin A and short methotrexate were used for graft-versus-host disease (GVHD) prophylaxis. After transplantation, all patients received 5 micrograms G-CSF/kg/d from day 1 until greater than 500 neutrophils/microL were reached and 150 U erythropoietin/kg/d from day 7 until erythrocyte transfusion independence for 7 days. Group I consisted of patients with acute myeloid leukemia (AML) (n = 2), chronic myeloid leukemia (CML) (n = 2), and T-gamma-lymphoproliferative syndrome and BM aplasia (n = 1). The patients received a mean of 3.3 x 10(6) CD34+ and 3.7 x 10(5) CD3+ cells/kg body weight of PBPC origin and 4.5 x 10(6) CD34+ and 172 x 10(5) cells/kg body weight of BM origin. Group II consisted of five patients (two AML, two CML, one non-Hodgkin's lymphoma). They received a mean of 3.3 x 10(6) CD34+ and 3.2 x 10(5) CD3+ cells/kg from PBPC and 1.4 x 10(6) CD34+ and 0.6 x 10(5) CD3+ cells from BM. A matched historical control group (n = 12) transplanted with a mean of 5.2 x 10(6) CD34+ and 156 x 10(5) CD3+ cells/kg from BM alone was assembled for comparison. In group I, the median time to neutrophil recovery to > 100, > 500, and > 1,000/microL was 12, 15, and 17 days, respectively. Patients from group II reached these neutrophil levels at days 13, 15 and 17 post BMT. Neutrophil recovery in the control patient group occurred at days 17, 18, and 20 respectively. Group I patients were given platelet transfusions within 18 days and red blood cells within 10 days, whereas for group II patients, these time points were 26 and 17 days, respectively. These same transfusions could be ceased within 38 and 24 days, respectively, in control patients. The addition of about 2% more peripheral blood CD3+ cells (group I patients) did not result in higher grades of acute GVHD (median grade II) as compared with the controls (median grade II). Four of five group II patients showed no signs of acute GVHD. These data suggest that the addition of immunoselected allogeneic CD34+ progenitor cells to BM cells may accelerate hematopoietic recovery.

Flow cytometry quantification of CD34+ cells and other leukocyte subpopulations in frozen-thawed blood cell suspensions: investigation of a new teflon container for cryopreservation of hematopoietic progenitor cells.

BACKGROUND: Cryopreservation is the only available method for the long-time maintenance of blood cells. The present study was designed to prove: (i) the reliability of multiparameter flow cytometry (MFC) for estimation of CD34+ cells in frozen-thawed cell suspensions and (ii) the acceptability of a new teflon container for the cryopreservation of hematopoietic progenitor cells. MATERIALS AND METHODS: Each of 15 ABO-compatible buffy coats (BC) were pooled, and mononuclear cells (MNC) were then separated with the Fresenius AS 104 device (n = 10). MNC harvested by apheresis were then divided into 2 portions and transferred pairwise into either the new Fresenius or into Gambro cryopreservation containers. Paired samples were frozen at controlled rates (9% DMSO final concentration) and stored at -196 degrees C in liquid nitrogen for 2 weeks. Leukocyte, MNC and differential blood counts and proportions of CD3+, CD4+, CD8+, CD14+ and CD34+ cells were assessed from the pooled BC, the apheresis products, and the frozen-thawed samples. Methyl cellulose culture assays as well as trypan blue viability staining were also carried out. RESULTS: The mean content of the divided apheresis products was 4.9 x 10(9) leukocytes with 86% MNC, 6.89 x 10(6) CD34+ cells, 2.1 x 10(5) granulocyte-macrophage colony-forming units (CFU-GM) and 7.1 x 10(5) erythroid burst-forming units (BFU-E). As expected, there were virtually no granulocytes after freezing in both types of container. The corresponding mean cell content was as follows: 6.3 x 10(6) CD34+ cells, 2.5 x 10(5) CFU-GM, and 8.1 x 10(5) BFU-E in Fresenius containers, and 6.1 x 10(6) CD34+ cells, 1.3 x 10(5) CFU-GM, and 7.7 x 10(5) BFU-E in Gambro containers. The mean MNC viability of the samples frozen in Fresenius was 81.5% and 82.7% in the Gambro containers. MFC was found to compare with stained smear differentials. CD34+ cell counts correlated with CFU-GM (0.69, p = 0.03) and BFU-E (0.63, p = 0.02) colony formation. CONCLUSIONS: The study reported here revealed no significant differences between the 2 types of storage containers. The new Fresenius teflon container could thus be recommended for cryopreservation of hematopoietic progenitor cells. MFC provided reliable data on CD34+ cell content and leukocyte subset composition of the frozen-thawed cell suspension.

[In-line-filtration of erythrocyte concentrates using the Sepacell Integra System]. / In-line-Filtration von Erythrozytenkonzentraten mittels Sepacell-Integra-System.

Using the Sepacell Integra System for in-line filtration of red cell concentrates in additive solution the leukocyte depletion was efficient enough to keep leukocyte contamination below the critical immunogenic load for leukocytes (CILL). The data indicate improved storage conditions, made evident by significantly lower cell damage.

Anticoagulation-dependent inhibition of in vitro complement activation by anti-apo-B sepharose 4B CL.

During LDL apheresis, various combinations of heparin and citrate are used for anticoagulation. With an in vitro batch system we examined whether heparin/citrate combinations can be optimized in terms of complement activation inhibition without the loss of anticoagulant potency. Plasma anticoagulated by using six clinically applicable regimens was incubated with anti-apo-B antibody-coupled Sepharose 4B CL, and the anaphylatoxin content of the supernatant was investigated. A significant dose-dependent reduction of complement activation was achieved by anticoagulating whole blood with 10 U/ml heparin (p < 0.05) if compared with serum whereas citrate inhibited more effectively the generation of C5a (desarg) even at a low concentration (ACD-B 1:20) (p < 0.01). The lowest anaphylatoxin level was generated when heparin (10 U/ml) plus citrate (ACD-B 1:10) were applied, although such an approach may be of limited clinical interest. The empirically chosen heparin plus citrate ratio (2 U/ml, 1:20, respectively) provides for an optimal and almost ideal inhibition of complement activation and contributes considerably to the good tolerability of the immunoadsorbent.

Anaphylatoxin generation and distribution during in vitro LDL apheresis.

The extent of anti-apo-B IgG-Sepharose-induced complement activation in serum and plasma (heparin 2 U/ml and ACD-B 1:20) was investigated using an in vitro model of LDL apheresis. The total volume of serum or plasma loaded to the chromatography column was collected in defined aliquots. The washing, desorption and regeneration fluids were processed in the same way. From the obtained values of generated complement split products C3a (desarg), C4a (desarg), C5a (desarg) and complement proteins C3, C4, C5, the conversion rates of the precursor were calculated. In the experiments with serum, 19% of C3, 8% of C4 and 2.3% of C5 were converted by the immunoadsorbent, whereas with plasma 7, 6, and 0.6%, respectively, were found. Furthermore, only 60-74% of total anaphylatoxins were found in the effluent during the loading process. The residual 26-40% was removed from the column with the subsequent washing fluids. Therefore, in the clinical routine, only a reduced part of generated anaphylatoxins will be retransfused to the patient. The fact that C5 is converted to the most limited extent to its biologically active fragment additionally contributes to the understanding of the good clinical tolerability of the LDL apheresis.

[Comparative evaluation of 3 leukocyte filters for thrombocyte concentrates]. / Vergleichende Evaluierung dreier Leukozytenfilter für Thrombozytenkonzentrate.

Several polyester adsorption filters are available for the removal of leukocytes during bedside filtration of platelet concentrates (PC). We tried to evaluate the efficacy of three leukocyte filters: PL-100 (Pall, USA), Sepacell PL-5A and PL-10A (both Asahi, Japan) using single donor PCs. All filters were used according to the manufacturer's instructions. The main difference in handling is the recommendation of priming and rinsing the Asahi filters with saline. Platelet counts were performed by an automatic cell counter, leukocyte (WBC) counts using a modified chamber method. Biocompatibility was examined by assessing anaphylatoxin, PMN-elastase and LDH levels, platelet function by measuring induced aggregation, hypotonic shock response, beta-thromboglobulin levels and the mean platelet volume. According to the different initial WBC contamination, three groups of PCs were tested. Group I and II were processed one hour after apheresis. Group III (n = 30) was stored at 12 degrees C up to five days before filtration. Function and integrity of post-filtration platelets were not affected as compared to the prefiltration status and bioincompatibility could not be detected. Our results indicate that the WBC reduction capacity of the filters is related to the initial WBC contamination of the PC. Beyond an initial contamination of 10(9) WBC, none of the filters is capable of reducing the leukocyte count below 10(6) WBC/PC. All but PL-5A reduce the WBC reliably below 10(6)/PC as determined from PCs routinely prepared. The Asahi filters cause significantly less platelet loss apparently due to the suggested postfiltration rinsing. Storage of PC does not influence the WBC reduction capacity, however increases the platelet loss for the PL-100.

Biocompatibility of extracorporeal immunoadsorption systems.

Following the first publication of a technique aiming for clinical application in 1969, extracorporeal immunoadsorption is increasingly finding a place among haemapheresis techniques. The apparent advantages outweigh the technical difficulties and economical drawbacks still connected with this treatment modality. At present three different developments are under clinical investigation: Staphylococcus Protein-A based immunoadsorption (SpA), the antibody-mediated adsorption (therapeutic affinity chromotography) and hydrophobic interaction-based immunoglobulin adsorption. The exact binding capacity and mechanism has not been completely elucidated for SpA and amino acid-based techniques. The lack of knowledge about pathogenic substrates exclusively responsible for the pathogenicity of many diseases, appears to be the major drawback for the prospective development of affinity chromatography-based extracorporeal systems. The practical application of molecular-biologic diagnostic procedures with a high grade of detection specificity (monoclonal antibodies) appears to be promising for the development of extracorporeal immunoadsorption. The immunomodulatory effect that results from the interaction of blood and plasma with artificial surfaces is an interesting subject of investigation, derived mainly from biocompatibility studies. The careful clinical and laboratory investigation of the biocompatibility of extracorporeal immunoadsorption systems contribute considerably to the prevention of undesired side effects.

Biocompatibility and safety of cytapheresis.

Cytaphereses are safe and can be used routinely like whole blood donations on the premise that standard techniques based on appropriate guidelines and regulations are performed. Before the introduction of a new cell or plasma separator, a thorough clinical investigation is always recommended in order to demonstrate biocompatibility and safety features. In this context cell loss and damage as well as activation of coagulation and complement should be tested primarily. The examination should consist of a direct comparison with an approved system. In addition, it is necessary to repeat periodically on multicenter studies adverse donor reactions such as those already described so that it is possible to recognize even rare side effects. Of special importance are long-term studies of donors undergoing frequent cytaphereses for many years in order to detect relevant immunological changes. On the other hand, harm caused by the plasticizer or cancer due to cancerogenicity of the plastics are only speculative at the moment.