Today, in addition to the classical test tube technology to determine red blood cell antigens and antibodies, not only the microtitration plate is increasingly used, but also the gel card. Since this new technology is easy to practice and since it further provides a higher specificity and sensitivity, we investigated whether, and if so to what extent, the gel card can be automated. On the basis of our own experience spanning many years, we were able to show that antigen determinations and antibody screening can be automated. Once the samples to be tested and the tests to be performed have been recorded by electronic data processing and combined in a job list, any sampler can distribute liquids, such as reagents and serum and/or plasma as well as red blood cell suspensions, on the basis of these data. A positive sample identification is made feasible by bar coding sample tubes and gel cards; the pipetting action is organized via the bar codes. Incubation and centrifugation are of secondary importance for automation. At this time, an automatic evaluation of gel cards is not yet possible so that result interpretation must be visual and the input of results must be manual. For documentation, however, laboratory books and result interpretation reports can be printed out automatically.
Peripheral blood stem cells are an alternative to bone marrow-harvested stem cells. We report on the feasibility of predicting optimal timing for leukapheresis by means of blood monitoring for CD34-positive cells by flow cytometry. In addition to monitoring, determinations of CD34-positive cells also are predictive for the hemopoietic potency of cells harvested by leukapheresis, as close correlations of numbers of CFU-GM and of CD34-positive cells, respectively, in leukapheresis samples are determined. Our data are indicating that flow-cytometrical determinations of CD34-positive cells are helpful in the setting of blood stem cell harvesting by leukapheresis, both for optimal timing of the procedure and for real-time estimation of the hemopoietic potency of cells harvested.
1 Originalbeitrag aus: Schleinzer W, Singbartl G (eds): Fremdblutsparende Maßnahmen in der operativen Medizin. Beitr Infusionsther. Basel, Karger, 1993, vol 29, pp 1 - 16. Mit freundlicher Genehmigung des Verlags.
The methods of collection and storage of peripheral blood stem cells are described and the harvesting after conditioning with cytokines and/or myelosuppressive therapy is discussed. It could be demonstrated that the combination of cytostatics and cytokines gives the best yield and enables to collect a sufficient amount of peripheral blood stem cells for a successful engraftment after myeloablative therapy with only few aphereses.
Four plasmapheresis procedures (manual blood bag centrifugation plasmapheresis, and the three plasmapheresis machines P.C.S./Haemonetics, Autopheresis-C/Baxter-Travenol, Plasmapur Monitor/Organon Teknika) were studied comparatively. The three machine procedures could be performed more easily and more rapidly and were well accepted by donors, autologous donors (patients) and staff. Compared with the traditional, well established manual procedure, a possible impairment of the plasma donors seems reduced rather than raised. Activation of the hemostatic system of the donors, measured with very sensitive methods, was found to be less pronounced when plasmapheresis was performed with the machines than when it was performed conventionally. The plasma product obtained by machine plasmapheresis was found to be of higher quality. All three systems showed less activation of the clotting system. Especially the plasma obtained by the P.C.S. showed a higher clotting factor yield. Plasma obtained by the Autopheresis-C and by the Plasmapur Monitor (both systems are equipped with filters) was markedly less contaminated with cells. The recently found low activation of the clotting system of plasma recipients, however, showed no differences when plasma obtained conventionally or by the Plasmapur Monitor was used. In summary, the new machine plasmapheresis devices offer a good alternative to the conventional blood bag centrifugation method and set new standards for the production of high quality plasma.
In addition to several conventional methods for the detection of red cell antigens, the use of microplates has various advantages either as a solid-phase assay (enzyme immunoassay) or as native microplate. Microplates may also be used for the detection of red cell antibodies in 'pooled-cell solid-phase assays' of the second generation and for antibody screening. Blood donors and patients are the two main fields which are to be examined in immunohematology. There are various advantages in using the microplate in blood group serology: (i) if there is hardware already available, like sample processors and microplate readers, the use of microplates in blood group serology reduces the costs even if the equipment has to be purchased for this purpose only; (ii) low quantities of reagents are used in microplate assays; (iii) the application of bar codes on tubes and microplates guarantees the most security in sample identification; (iv) it is possible to investigate blood samples selectively depending on the available software if antibody detection is done as the sixth test beside anti-HIV, anti-HCV, HBsAG, lues antibodies and ALT, and (v) recording of data will be easy if electronic data processing is used.
The human factor VIII gene and the human von Willebrand factor gene have been isolated, and were transfected into a mammalian cell line (Chinese Hamster Ovary Cells = CHO Cells). The resulting factor VIII producing cell line was thoroughly characterized. No differences were found in the recombinant factor VIII compared to plasma-derived factor VIII. For factor VIII production the cells are grown in serum-free defined medium, the recombinant factor VIII is purified by immunoaffinity chromatography plus two subsequent ion exchange steps. The absence of human raw materials for production, and a proven titer reduction of > 10(7) in the purification process significantly minimize the risk of virus transmission by the recombinant factor VIII product. Clinical studies both with previously treated and untreated patients have proven the good hemostatic efficacy and virus safety of the recombinant factor VIII. The inhibitor incidence in previously untreated patients is within the expected range for this patient group.
To improve the separation results of peripheral blood stem cells (PBSC), cytokines (GM-CSF, G-CSF or IL3) can be administered to patients. The most important prerequisites for successful PBSC separation are daily monitoring of CD-34-positive cells to determine the separation days and the use of optimized separation programs. To improve the performance of the cell separation, we increased the process volume to more than patients' blood volume and obtained increased MNC and CFU recoveries. In most cases, we collected more cells than the preseparation number of circulating cells. We therefore conclude that large-volume PBSC separation itself mobilizes hematopoietic progenitor cells.
Transfusion of blood and blood components may be fraught with serious immunologically mediated side effects. Acute hemolytic reactions are still the most common cause of fatal transfusion sequelae. The incidence of alloimmunization against erythrocyte antigens as studied in long-term transfused patients with thalassemia depends on the age at the beginning of transfusion therapy. HLA alloimmunization is often associated with refractoriness to platelet transfusions and febrile transfusion reactions. Neonatal alloimmune thrombocytopenia and post-transfusion purpura are elicited by platelet-specific antibodies reacting with determinants on platelet glycoproteins IIb/IIIa, Ib/IX, and Ia/IIa. Another serious complication of transfusion therapy, transfusion-related acute lung injury, is caused by granulocyte-specific alloantigens in donor plasma. Graft-versus-host disease is a rare but dangerous complication of blood transfusion which mainly affects patients with impaired T-cell-related immunity.
In patients with acute leukemia, chemotherapy leads to subsequent aplasia-associated thrombocytopenia requiring the substitution of platelets to avoid and treat bleeding complications. A multitude of tests to assess the quality of platelet concentrates is available to date. In this study, we investigated the quality of platelet concentrates prepared by two different methods, preparation from platelet-rich plasma versus preparation from buffy coat, and stored under varying conditions, namely horizontal versus vertical rotation, by measuring the binding rate of corresponding antibodies to platelet membrane glycoproteins GPIb, GPIIb-IIIa, GPIa-IIa and GPIV using flow cytometry. Expression of platelet surface antigens was well maintained during platelet preparation and storage for 7 days. Preparation and storage modalities showed no significant effect on the expression of membrane glycoproteins. These results indicate that platelet function as represented by antibody binding is well maintained during platelet preparation and storage leading to sufficient hemostasis following transfusion.
We report on the adaptation of a 'Walkaway' instrument (ES 600, Boehringer Mannheim) to be used in a blood transfusion service for donor screening of HBsAG and anti-HIV 1 + 2. Anti-CMV is obtainable, and anti-HCV will be available in the near future. Sample processing is done by a Tecan 8051 ID from barcoded primary tubes into ES 600 carousels. The capacity of this instrument is limited to 150 specimens and 600 tests per run. After mounting of reagents and control specimens, the tests are performed fully automatically without a technician. Regarding our experiences, the instrument is unanimously suited for use of blood donor testing in transfusion services with less than 145 samples to be tested daily.
Quality controls of autologous blood collections in critically ill patients comprise the control of blood products, blood collection, and of the patients themselves. The control of products is defined in European guidelines, the AMG (law governing the manufacture and prescription of medicine) and GMP regulations. The products are described in the monograph of the Federal Health Office. The quality control of blood collection in patients with a critical vascular disease is important since vagotonic or hypertensive crises may occur frequently (in 10-15% of cardiosurgical patients). The quality control of the critically ill patients themselves is important in order to be able to balance benefits against risks. A phlebotomy of 500 ml may lead to a considerable deterioration of the clinical condition. The clinical condition can be controlled by simple exercise tests prior to and after the blood collection (bicycle ergometer, treadmill or climbing stairs). In our own investigations only about 25% of cardiosurgical patients (40% of patients with aortocoronary venous bypass) received autohemotherapy, and 20% of them showed a clinical deterioration during the phase of blood collection. Other problematic patients are those suffering from a tumor. A clear clinical benefit of autohemotherapy in these patients has not been demonstrated up to now; nevertheless, when a curative therapy is possible, they should be treated with autohemotherapy.
Fresh plasma from single-blood donations is virus-inactivated by illuminating the plasma units in their respective plastic bags with visible light for 1 h in the presence of 1 microM of the photoactive dye methylene blue. For "soft" viruses, e.g. the togavirus Semliki Forest, 0.3 microM are sufficient to achieve complete inactivation within 5-10 min. The infectious titer is reduced by 5-6 log 10 steps. To achieve the same degree of reduction in infectivity for more resistant viruses, e.g. vesicular stomatitis virus or SV 40, the virus-containing plasma at a dye concentration of 1 microM has to be light-treated for 30-45 min. More time is required at lower dye concentrations. These findings determined the conditions chosen for photodynamic virus inactivation of fresh plasma from single-blood donations.
When no specific factor concentrate is available fresh-frozen plasma (FFP) is indicated in the treatment of clinically relevant hemorrhagic diathesis. These disorders include congenital factor V and XI deficiencies, multiple factor defects, as disseminated intravascular coagulation and severe liver disease, and patients receiving massive transfusions, when bleeding occurs and severe abnormalities on coagulation testing are evident. FFP is beneficial when used with plasma exchange in thrombotic thrombocytopenic purpura and related disorders. Various virucidal treatments including solvent-detergent (SD), photoactivated dyes (methylene blue) or pasteurization have been evolved to improve virus safety of human plasma. More extensive studies to demonstrate efficient virus inactivation in plasma have been performed with SD compared to other methods. On the other hand, the use of single-donor FFP in methylene blue treatment is possibly superior to pooled plasma which is processed according to the SD procedure. Pasteurization enables the inactivation not only of lipid-enveloped but also of non-lipid-enveloped viruses. Virucidal treatment of plasma may cause alterations in clotting factors, fibrinolysis and protease inhibitors; however, the currently achieved recovery of procoagulant activities is approximately comparable with that found in untreated FFP. The toxicity of virucidal additives is reported to be negligible since manufacturing includes a removal procedure (SD) or comparably low amounts (methylene blue) are used in inactivation treatment.
Modern haemotherapy is equivalent to restrictive use of blood components. Therefore, transfusion of whole blood in homologous transfusion generally cannot be accepted. In autologous blood transfusion blood components also are preferable if they can be separated appropriately. In order to have broad application of preoperative autologous blood deposits, close cooperation to transfusion services should be established guaranteeing optimal production of blood components. If this cooperation is impossible there are no objections against the use of autologous whole blood as long as the expected blood consumption is less than 3 red cell units. Additionally, in this case storage of more than 3 weeks mostly is not necessary. The fact that whole blood is not further part of the 'Monographien' of the Federal Health Administration (BGA) does not forbid the use of autologous whole blood since the 'Monographien' only concern generally available homologous blood components.
Today, in addition to the classical test tube technology to determine red blood cell antigens and antibodies, not only the microtitration plate is increasingly used, but also the gel card. Since this new technology is easy to practice and since it further provides a higher specificity and sensitivity, we investigated whether, and if so to what extent, the gel card can be automated. On the basis of our own experience spanning many years, we were able to show that antigen determinations and antibody screening can be automated. Once the samples to be tested and the tests to be performed have been recorded by electronic data processing and combined in a job list, any sampler can distribute liquids, such as reagents and serum and/or plasma as well as red blood cell suspensions, on the basis of these data. A positive sample identification is made feasible by bar coding sample tubes and gel cards; the pipetting action is organized via the bar codes. Incubation and centrifugation are of secondary importance for automation. At this time, an automatic evaluation of gel cards is not yet possible so that result interpretation must be visual and the input of results must be manual. For documentation, however, laboratory books and result interpretation reports can be printed out automatically.
Since 1992, the Fresenius cell separator AS-104 offers a single-needle option. The authors present data on their first experience with this new single-needle thrombocytapheresis system.
