The isotype of autoantibodies influences the phagocytosis of antibody-coated platelets in autoimmune thrombocytopenic purpura.

Autoimmune thrombocytopenic purpura (AITP) is an acquired autoimmune bleeding disorder, characterized by isolated thrombocytopenia because of destruction of auto-antibody-coated platelets by Fc-receptor-mediated phagocytosis. The destruction of autoantibody-sensitized platelets by FcγR-bearing phagocytic cells and the following antigen presentation are considered to play a key role for the pathophysiology of AITP. Although different isotypes of AITP-mediating autoantibodies, e.g. IgG, IgM and IgA, are frequently found in AITP patients, their role in the pathophysiology of AITP remains unclear. Using a flow cytometric monocyte-based phagocytosis assay, we investigated the impact of disease-associated autoantibody isotype in antibody-mediated phagocytosis of platelets. Platelets, labelled with 5-chloromethyl fluorescein diacetate (CMFDA), were incubated with AITP patients' serum characterized by pure IgG or IgM antiplatelet autoantibodies. Labelled platelets were incubated with monocytes. Phagocytosis was defined as the product of percentage of CMFDA-positive monocytes and mean fluorescence intensity of CMFDA. Adherence of platelets to monocytes was quantified by anti-CD61-PerCp in a CMFDA(+) CD14(+) gate. IgG-coated platelets showed a significantly higher phagocytic index than IgM-coated platelets (mean 796 ± 157 versus 539 ± 78, P < 0.01). There were no significant differences regarding platelet adherence to monocytes. The isotype of autoantibodies influences the quantity of in vitro phagocytosis of autologous platelets by monocytes. Therefore, the AITP-mediating autoantibody isotype should be considered more carefully in pathophysiologic models and furthermore in diagnostic, therapeutic and prognostic approaches in AITP.

[Risks and side effects of blood transfusion]. / Risiken und Nebenwirkungen von Bluttransfusionen.

While transfusion of blood components is usually safe, there are risks of adverse effects that can have immunologic, nonimmunologic, or infectious causes. In patients, the fear of infectious disease transmission predominates, although the risk has been extremely low since the introduction of reliable serologic and molecular biological testing methods. This article addresses the incidence, clinical picture, and etiology of adverse effects of transfusion. It also reports on current knowledge concerning transfusion-associated acute lung injury, which has gained much attention in the last few years. Besides hepatitis and human immunodeficiency viruses, cytomegalovirus, parvovirus B19, prion transmission, and the risk of variant Creutzfeld-Jakob disease are also discussed.

Detection of bacteria in platelet concentrates prepared from spiked single donations using cultural and molecular genetic methods.

Bacteria show differences in their growth kinetics depending on the type of blood component. On to storage at 22 degrees C, platelet concentrates (PCs) seem to be more prone to bacterial multiplication than red cell concentrates. Knowledge of the potential for bacterial proliferation in blood components, which are stored at a range of temperatures, is essential before considering implementation of a detection strategy. The efficacy of bacterial detection was determined, using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), following bacterial growth in blood components obtained from a deliberately contaminated whole-blood (WB) unit. Cultivation was used as the reference method. WB was spiked with 2 colony-forming units mL(-1)Staphylococcus epidermidis or Klebsiella pneumoniae, kept for 15 h at room temperature and component preparation was processed. Samples were drawn, at intervals throughout the whole separation process, from each blood component. Nucleic acids were extracted using an automated high-volume extraction method. The 15-h storage revealed an insignificant increase in bacterial titre. No bacterial growth was detected in red blood cell or plasma units. K. pneumoniae showed rapid growth in the pooled PC and could be detected immediately after preparation using RT-PCR. S. epidermidis grew slowly and was detected 24 h after separation. These experiments show that sampling is indicative at 24 h after preparation of PCs at the earliest to minimize the sampling error.

Quantification of PCR products using microparticles and flow cytometry.

The analysis of genetic biomarkers has become an important tool in clinical diagnostics. This includes the identification of disease-related genetic alterations, the detection of pathogenic infective germs on DNA or RNA level and the quantification of the expression of marker genes indicating an altered physiological status. It has been previously described that the combination of polymerase chain reaction (PCR), microparticles and flow cytometry represents a universal platform technology for the routine analysis of such biomarkers. Here we demonstrate the applicability and flexibility of this technology by means of various applications. The quantification of interferon gamma (IFNG) mRNA in irradiated white blood cells is shown as well as the detection of latent infections with cytomegalovirus (CMV). Besides the quantification of single amplification products, the flow cytometric assay is also capable of analysing products of a multiplex PCR. As an example, we describe the identification of spontaneous deletions in the genome of a hybrid cell line using a co-amplified gene (RAB1) essential for the cell survival as an internal control. Furthermore, we show that the use of a green laser (532 nm, 50 mW) substantially increased the sensitivity of the assay compared to conventional flow cytometers using a 488 nm (25 mW) laser. We conclude that the analysis of PCR products using microparticles and flow cytometry fulfils the criteria of clinical routine diagnostics regarding (i) sensitivity, (ii) specificity, (iii) reproducibility and (iv) automatibility.

Tolerance of granulocyte donors towards granulocyte colony-stimulating factor stimulation and of patients towards granulocyte transfusions: results of a multicentre study.

Data of 507 granulocyte donations from 183 donors were evaluated. No severe granulocyte colony-stimulating factor (G-CSF)-related side-effects were observed. Three donors complained of severe itching following infusion of hydroxyethyl starch (HES). A high proportion (85%) of the donors stated that they would donate granulocytes again. The mean granulocyte yield was 4.3 x 10(10). High-molecular-weight HES resulted in a significantly higher yield compared with low-molecular-weight HES. Mild, but no severe, adverse transfusion reactions were observed in 16% of the recipients. A leucocyte alloimmunization rate of 24% was found. G-CSF stimulation and transfusion of G-CSF-mobilized granulocytes were well tolerated by donors and recipients, respectively.

Autologous red cells derived from cord blood: collection, preparation, storage and quality controls with optimal additive storage medium (Sag-mannitol).

To investigate whether packed red cells (PRCs) prepared from autologous cord blood-packed red cells (AC-PRCs) could be used as an alternative for homologous-packed red cells (H-PRCs), we developed a system to collect and prepare AC-PRCs and determined standard storage parameters during 35 days of storage in extended storage medium (Sag-mannitol). We collected and fractionated cord blood from 390 newborns. The amount and quality of the AC-PRCs were analysed. The bacterial contamination rate was 1.84%. Twelve AC-PRCs were stored for 35 days, and standard laboratory parameters were measured at day 1 and day 35. The initial laboratory parameters of the AC-PRCs were similar to the parameters of the H-PRCs. After 35 days, the AC-PRCs displayed an increased haemolysis rate compared to H-PRCs (1.1 versus 0.2%) and also a significant decreased adenosine triphosphate value (1.2 versus 2.3 micromol L(-1)). Haemoglobin, haematocrit and pH were comparable in both groups. AC-PRCs meet the quality criteria for H-PRCs after 35 days. Utilizing a closed collection system for cord blood and an extended storage medium will increase safety and quality and facilitate the routine transfusion of autologous red cells derived from cord blood.

Strategies to reduce perioperative blood loss related to non-surgical bleeding.

The treatment of critically ill patients has advanced markedly over the last decade. However, non-surgical bleeding of a diffuse nature from numerous tiny capillaries still remains a challenge. Once initiated, this type of bleeding may be troublesome and a vicious circle develops since it is not a single vessel contributing to this blood loss. The description 'non-surgical blood loss' is often given to this. This review describes a step-by-step approach for the treatment of non-surgical bleeding and includes various measures, such as desmopressin, blood components, antifibrinolytics, antithrombin III, prothrombin complex concentrates and factor XIII. While most non-surgical bleedings can be managed using the approach described here, a number of patients still continue to bleed. In these cases, the surgeon should re-evaluate the bleeding in terms of its surgical origin. If this can positively be excluded and if all of measures described fail to reduce or stop the bleeding, further treatment of such uncontrolled bleeding remains symptomatic.

A novel true volumetric method for the determination of residual leucocytes in blood components.

BACKGROUND AND OBJECTIVES: Accurate determination of residual leucocytes [white blood cells (WBC)] in blood components is of high clinical importance. To date, several labour-intensive, time-consuming or expensive techniques have been used for this purpose. MATERIALS AND METHODS: A method for the determination of residual WBC is described using a novel low-cost flow-cytometric cell counter and analyser (CCA). The DNA in WBC was stained using 4'-6-diamidino-2-phenylindole (DAPI) and WBC were automatically analysed by true volumetric counting of 200-microl samples (prepared from a 20-microl undiluted sample). RESULTS: Dilution experiments over a range of 0.5-50 WBC/microl showed a linearity of r = 0.998. The detection limit of this method was 0.83 WBC/microl of red blood cell concentrate (RCC) and 0.67 WBC/microl of platelet concentrate (PC), with an accuracy of 95.5%. CONCLUSION: Residual WBC (< 1 WBC/microl) can be accurately counted using the CCA within 2 min and at a total cost of less than euro 1 per sample.

Kinetics of standardized large volume leukapheresis (LVL) in patients do not show a recruitment phenomenon of peripheral blood progenitor cells (PBPC).

Although several studies have demonstrated the efficacy of large volume leukapheresis (LVL) to yield high numbers of peripheral blood progenitor cells (PBPC), the mechanisms of stem cell release into circulation and the postulated phenomenon of PBPC recruitment during apheresis have not been investigated in detail. Therefore, we analyzed the kinetics of stem cell enrichment in a total of 34 standardized LVL for patients with hematologic malignancies (lymphoma, multiple myeloma) and solid tumors (breast cancer, rhabdomyosarcoma). LVL was started 2 h after administration of G-CSF processing six times the patient's blood volume. Cells were sequentially collected into six bags and the numbers of leukocytes, mononuclear cells (MNC), CD34+ cells and colony-forming cells (CFU-GM) in each collection bag were analyzed. The numbers of PBPC collected demonstrated a continuous decrease starting after an early maximum during the second processed blood volume (P = 0.001). Interestingly, these kinetics of decreasing stem cell yields during LVL were similar for both entities of patients with hematologic malignancies as well as for both groups of patients with solid tumors. In summary, a recruitment phenomenon, defined as a time-dependent and LVL-induced increase of PBPC, could not be demonstrated in any of the diseases investigated.

Automated serological compatibility testing using a solid-phase test and standard laboratory equipment.

BACKGROUND AND OBJECTIVES: To prove the feasibility of a semi-automated cross-match procedure using a commercially available solid-phase microplate test and standard laboratory equipment. MATERIAL AND METHODS: The new procedure was evaluated against the conventional spin tube technique and the gel centrifugation system. RESULTS: The sensitivity of the method and the rate of non-specific reactions were equal to those for the other test systems. The samples taken from the red cell concentrates for cross-matching remained stable for the shelf-life of the product. CONCLUSION: The semi-automated cross-match was successfully introduced in our routine laboratory as a means to process large numbers of tests.

Polymorphisms in the non-coding region of the human mitochondrial genome in unrelated plateletapheresis donors.

Human mitochondrial DNA polymorphisms are unique targets to discriminate nucleated cells and platelets between donor and recipient in the setting of transplantation or transfusion. We have previously used this approach to discriminate allogeneic platelets from autologous platelets after transfusion. In the present study, we used DNA sequencing to investigate polymorphisms present in two of the hypervariable segments (HVR1 and HVR2) found within the non-coding region of the mitochondrial genome among 100 plateletapheresis donors. Alignments were made with the Cambridge Reference Sequence (CRS) for human mitochondrial DNA (mtDNA). Combining the sequencing information of HVR1 and HVR2 we could demonstrate that, of the 100 investigated mtDNA samples, none was identical to the CRS. We found a total of 2-17 polymorphisms per donor in the investigated regions, most of them were basepair substitutions (563) and insertions (151). No deletions were found. Sixty-six of the 110 detected polymorphisms were detected in more than one sample. Seven polymorphisms are newly described and have not been published in the Mitomap database. Our results demonstrate that polymerase chain reaction analysis of the many polymorphisms found in the hypervariable region of mitochondrial DNA represents a more informative target than previously described mitochondrial polymorphisms for discriminating donor-recipient cells after transfusion or transplantation.

Simultaneous immunomagnetic CD34+ cell selection and B-cell depletion in peripheral blood progenitor cell samples of patients suffering from B-cell non-Hodgkin's lymphoma.

The reduction of residual tumor cells is one of the main targets of leukapheresis product (LP) processing. Immunomagnetic enrichment/selection of CD34+ progenitor cells (Baxter Isolex 300i) can achieve a reduction of contaminating B-cells of approximately 2-3 logs in B-cell non-Hodgkin's lymphoma patients. Specific release of the enriched CD34+ cells (stem cell releasing agent PR34+; Baxter) and the use of antibody-coated immunobeads targeted against B-cell markers (CD10, CD19, CD20, CD22, CD23, and CD37) during this procedure allows the GMP-like simultaneous capture of residual B cells within a closed system. This combination of two purging techniques enhances the B-cell depletion capacity up to 4.5 logs. By performing 10 clinical-scale purging procedures, we could show that the simultaneous immunomagnetic purging method is easy to perform and highly efficient. We evaluated B-cell log depletion by flow cytometry for cases with marker-positive cells detectable before and after the purging procedure. The mean reduction of B-cells in these cases was 3.5 logs; the mean CD34+ cell yield and purity were 47 and 92%. Using three LPs, we tested the procedure on a modified Baxter Isolex 300i device with software adaptations for this procedure (software version 2.0) in direct comparison with CD34+ cell selection only, using the former version (version 1.12). The CD34+ cell yield was 49% (40-54%) for the CD34+ cell selection and 51% (19-72%) for simultaneous double selection. The mean purity was 96% for CD34+ cell selection and 98% for simultaneous double selection. B-cell depletion was 1.9 logs for CD34+ cell selection, and after simultaneous double selection, the B-cell content was decreased by 3.7 log steps (P = 0.0495). Clinical application of double-purged cells has not prolonged the hematopoietic recovery times after high-dose therapy as compared with nonpurged peripheral blood progenitor cell autotransplants. In conclusion, we could show that the simultaneous double selection protocol developed leads to a highly increased B-cell purging efficacy when compared with CD34+ cell selection without any negative effects regarding CD34+ cell yield and engraftment times after high-dose therapy.

Platelet glycoprotein complex Ia/IIa antibodies cause neonatal alloimmune thrombocytopenia but do not inhibit megakaryopoiesis and platelet recovery after allogeneic cord blood stem cell transplantation.

A sibling cord blood (CB) transplantation was performed in a boy with Wiskott-Aldrich syndrome. The CB (31 x 10(6) CD34(+) cells) derived from a newborn sister with neonatal alloimmune thrombocytopenia (NAIT) with 40,000 platelets/microl, caused by a maternal anti-HPA-5b and HLA-A2 antibody. Maternal serum did not inhibit clonogenicity after in vitro testing of megakaryopoiesis. Accordingly, this CB was accepted for sibling transplantation. The transplantation showed a good course with fast and sustained hematopoietic reconstitution (granulocytes >500/microl on day +16, platelets >50,000/microl on day +30). This case demonstrates a successful CB transplantation from a donor suffering from NAIT.

HLA-antibody testing: the immune phagocytosis inhibition test is superior to the PRA-STAT and NIH lymphocytotoxic test with respect to specificity.

We compared the specificity and sensitivity of four different methods for the detection of antibodies specific for HLA antigens. The NIH version of the complement-dependent cytotoxic test (CDC) was used as the gold standard to which we compared two Fcgamma receptor (FcgammaR)-dependent immune phagocytosis inhibition tests (IPI) and one commercial enzyme-labelled immunosorbent assay (ELISA) with soluble HLA class I-antigen preparations bound to the plate (PRA-STAT). Both IPI tests are based on the fact that HLA-antibodies specifically bind to antigens on the monocyte surface via their Fab portion, and in so doing block a neighbouring FcgammaR with their Fc region. This blockade prevents phagocytosis of IgG-coated red blood cells (RBCs), which can be measured either microscopically (IPIm) or photometrically (IPIp). The four assays were used in blind tests on 20 human alloantisera or monoclonal antibodies with known HLA-antigen reactivities. Additionally, two monoclonal antibodies and one human serum were titrated to elucidate the sensitivity of each test. After all tests were completed, the identities of the samples were disclosed. Both IPI methods detected and identified all clinically relevant HLA class I and class II specific antibodies. In contrast, the CDC was not able to detect noncytotoxic HLA-antibodies and HLA class II specific antibodies; however, it detected clinically insignificant IgM lymphocytotoxins. The PRA-STAT assay enabled identification of all cytotoxic and noncytotoxic IgG antibodies with specificity for HLA-class I antigens. With respect to sensitivity, the CDC and the IPI methods were superior to the PRA-STAT. These facts demonstrate the advantage of IPI methods in the detection of clinically relevant HLA-antibodies.

Volume-dependent collection of peripheral blood progenitor cells during large-volume leukapheresis for patients with solid tumours and haematological malignancies.

We investigated the efficacy of peripheral blood progenitor cell (PBPC) collection during large-volume leukapheresis (LVL) in patients with solid tumours and haematological malignancies (n = 18). The time- and volume-dependent harvest of leucocytes (WBC), mononuclear cells (MNC), CD34+ cells and colony-forming cells (CFU-GM) during LVL was analysed in six sequentially filled collection bags processing four times the patient's blood volumes. The amounts of leucocytes (WBC) and the purity of mononuclear cells (MNC%) did not show any significant changes during LVL. The percentage of CD34+ cells remained constant for the first three bags but consecutively decreased from initially 1.71% CD34+ cells in the beginning of LVL to finally 1.34% CD34+ cells (P = 0.02). The mean numbers of colony-forming cells (CFU-GM) decreased from 74 microL-1 to 59 microL-1 during LVL (P = 0.16). Furthermore, the comparison of volume-dependent PBPC collection for patients with high, medium and low total yields of CD34+ cells showed similar kinetics on different levels for the three groups. We concluded that - relative to the initial total amount of PBPC harvested - comparable numbers of progenitor cells can be collected during all stages of LVL with a slight decreasing trend processing four times the patient's blood volumes.

Efficacy and safety of simultaneous immunomagnetic CD34+ cell selection and breast cancer cell purging in peripheral blood progenitor cell samples used for hematopoietic rescue after high-dose therapy.

We have established a new simultaneous positive/negative selection procedure using the Baxter Isolex 300i system. We tested its tumor cell (TC) purging efficacy by tumor contamination tests ex vivo and its safety in a group of 17 breast cancer (BC) patients by measuring hematopoietic recovery after high-dose (HD) therapy and autologous stem cell rescue with the selected cells. Tumor contamination tests resulted in a TC depletion of 4.1-6.0 log steps. The CD34+ cell yield in this experimental setting was 38.9-91.5%, and the CD34+ cell purity was 86.0-96.0%. In a group of 17 BC patients (5 high-risk adjuvant, > or = 10 lymph nodes positive, and 12 metastatic), we processed leukapheresis products (LPs) by simultaneous positive/negative selection. In these clinical samples, the mean CD34+ cell yield was 56.2% (range, 14.0-80.1%), and the CD34+ cell purity was 94.5% (range, 69.0-99.8%). Additionally, we screened samples of the patients' LPs before and after the purging procedure for contaminating TC by immunocytochemistry. In 15 of 17 tested cases, TCs were detectable prior to the purging procedure. After the procedure, we could not detect residual TCs in 16 of 17 cases. In one case, we found a highly reduced number of TCs. Furthermore, we evaluated the times for hematopoietic reconstitution in a group of five BC patients in the high-risk adjuvant situation who underwent HD chemotherapy and hematopoietic rescue with positive/negative selected stem cells and compared it with our own data from 10 BC patients who, after identical HD therapy, received only positively selected CD34+ cells and 14 patients who, after identical HD therapy, received autografts purged by incubation with toxic ether lipids (ET-18-OCH3). In all groups, a leukocyte count of >2000 cells/microl was reached at day +10. A platelet count of > 50,000 cells/microl was reached at day +12 in the ET-18-OCH3 group and at day +14 in the other two groups. Furthermore, 12 patients with metastatic disease rescued with positive/negative selected stem cells after HD therapy also showed fast and comparable hematopoietic recovery. The new simultaneous immunomagnetic positive/negative selection using a closed system is effective and safe. Processing LPs leads to a similar CD34+ cell yield, a higher TC depletion compared to standard CD34+ cell selection, and no delay in hematopoietic recovery.

[Peripheral blood stem cell transplantation as an interdisciplinary challenge--theory and practice]. / Die periphere Blutstammzelltransplantation als interdisziplinäre Herausforderung--Theorie und Praxis.

High dose chemotherapy with consecutive autologous peripheral blood stem cell transplantation becomes increasingly important for the treatment of hematological diseases and solid tumors. A complete remission or at least a prolonged survival can be achieved for numerous malignant diseases by an intensification of chemo- and radiotherapy. Therefore, the autologous peripheral blood stem cell transplantation (PBSCT) represents an elementary precaution to reduce the therapy-associated aplasia by administration of hematopoietic precursor cells. Both, high dose chemotherapy with consecutive PBSCT demands great clinical experience and the collection, processing and positive selection of blood stem cells is a challenge for the Transfusion Medicine. Correct handling and utilization of blood stem cells for clinical and laboratory purposes (e.g. positive selection) must be guaranteed, since each restriction of the function of processed blood stem cells may lead to an insufficient engraftment after PBSCT. Therefore, the clinical divisions of the University Hospital Münster are planning and practising peripheral blood stem cell transplantations in cooperation with the Department of Transfusion Medicine. The collection, processing and quality control are performed by the Department of Transfusion Medicine in close contact with the other clinical departments, who subsequently perform high dose chemotherapy and peripheral blood stem cell transplantations.

Suppression of panel-reactive antibodies by treatment with mycophenolate mofetil.

"Panel Reactive Antibody" (PRA) testing is commonly used to assess the pretransplant antibody status in order to estimate the risk of an adverse humoral response following transplantation. We report on a female patient with end-stage cardiac failure suffering from acute myocarditis who underwent implantation of a left-ventricular assist device (Novacor, Baxter Healthcare Corp. Oakland, CA). During evaluation for heart transplantation, a PRA level of 50-70% was detected. After treatment with mycophenolate mofetil at a dosage of 2 g daily, PRA levels declined within one week to 0-5%, and remained low after discontinuation of the immunosuppressive drug. We feel that pretreatment of patients with elevated PRA levels with mycophenolate mofetil is well justified.

Flow cytometric analysis of protein phosphorylation in the hematopoetic system.

Cellular growth and differentiation in blood cells are regulated by the phosphorylation status of growth factor receptors and downstream proteins. Protein kinases and phosphatases balance the homeostasis of protein phosphorylation. Various diseases are associated with alterations in these tightly regulated processes. Aberrations have been proved to be of diagnostic value and might enhance the pathophysiological insight into the origin of the disease. However, quantitation of protein phosphorylation is currently not feasible in a clinical situation. We developed a flow cytometric methodology which enables for direct investigation of protein phosphorylation in cell populations defined by multi-color flow cytometry. This assay does not only overcome drawbacks of traditional methodologies (e.g. Western blotting) but also allows quantitative analyses even in rare cell populations. We accurately examined phosphorylation levels in different cell populations of hematological interest and especially analyzed CD34+ hematopoietic progenitor cells. CD34+ cells in bone marrow and in cord blood contained similar, low levels of phosphotyrosine. Circulating pheripheral blood system cells PBSC in patients exposed to G-CSF for stem cell mobilization exhibited significantly increased levels of phosphotyrosine. In vitro exposure of CD34+ progenitors to growth factors (G-CSF, IL-3, SCF) raised the levels of tyrosine phosphorylation in bone marrow and cord blood. Effects were dose and time dependent. Interestingly, in vivo stimulated CD34+ PBSC could not be further stimulated in vitro. In conclusion, we present a new powerful methodology for analysis of protein phosphorylation in hematological specimens. The method does not only allow for accurate detection of phosphorylation levels in vivo, but also enables for quantitative analysis of growth factor receptor stimulation in vitro and in vivo.