Nano-sized and micro-sized polystyrene particles affect phagocyte function.

Adverse effect of nanoparticles may include impairment of phagocyte function. To identify the effect of nanoparticle size on uptake, cytotoxicity, chemotaxis, cytokine secretion, phagocytosis, oxidative burst, nitric oxide production and myeloperoxidase release, leukocytes isolated from human peripheral blood, monocytes and macrophages were studied. Carboxyl polystyrene (CPS) particles in sizes between 20 and 1,000 nm served as model particles. Twenty nanometers CPS particles were taken up passively, while larger CPS particles entered cells actively and passively. Twenty nanometers CPS were cytotoxic to all phagocytes, ≥500 nm CPS particles only to macrophages. Twenty nanometers CPS particles stimulated IL-8 secretion in human monocytes and induced oxidative burst in monocytes. Five hundred nanometers and 1,000 nm CPS particles stimulated IL-6 and IL-8 secretion in monocytes and macrophages, chemotaxis towards a chemotactic stimulus of monocytes and phagocytosis of bacteria by macrophages and provoked an oxidative burst of granulocytes. At very high concentrations, CPS particles of 20 and 500 nm stimulated myeloperoxidase release of granulocytes and nitric oxide generation in macrophages. Cytotoxic effect could contribute to some of the observed effects. In the absence of cytotoxicity, 500 and 1,000 nm CPS particles appear to influence phagocyte function to a greater extent than particles in other sizes.

Influence of multicomponent apheresis on donors' haematological and coagulation parameters, iron storage and platelet function.

BACKGROUND AND OBJECTIVES: Multicomponent collection (MCC) enables production and processing of various blood components during one apheresis session. In this prospective crossover study, the effects of donating platelets (PLTs) and packed red blood cells (PRBCs) on donor's blood cell count, coagulation, PLT function and iron state were analysed. MATERIALS AND METHODS: Forty-eight MCCs were performed using two different cell separators (Fenwal Amicus(®), CaridianBCT Trima Accel(®)). Two units of platelet concentrates and one unit of PRBCs were collected during each session. Full blood cell count and iron status were obtained on day 0 before and after apheresis, day 2, day 14 and day 42. PLT function was analysed by aggregometry and rotation thromboelastometry in parallel with coagulation tests before and after MCC and at day 2. RESULTS: Multicomponent collection was well tolerated without adverse side effects. Blood cell count and iron parameters declined and most of them (haemoglobin, haematocrit, transferrin, transferrin saturation and ferritin) were significantly below baseline values until at least day 42 after donation. Absent iron stores were seen in 31·3% of the donors. In contrast, PLTs significantly exceeded pre-donation values after 14 days and remained significantly increased for 42 days. After 2 days, coagulation parameters were only slightly (P > 0·05) altered, whereas PLT function was significantly reduced. CONCLUSION: Multicomponent collection is an obviously safe procedure; however, the significant long-term impact on the donor's blood count and iron store, as well as impaired PLT function, has to be considered in regard to donor safety.

Reticulocyte hemoglobin content allows early and reliable detection of functional iron deficiency in blood donors.

BACKGROUND: Frequent blood donations may lead to a depletion of body iron stores resulting in manifest anemia. Reticulocyte hemoglobin content (CHr) - a marker for impaired hemoglobinisation (IH) caused by functional iron deficiency (FID) - was investigated regarding its value as a routine screening parameter in frequent whole blood donors. METHODS: In a prospective study, 917 frequent blood donors and 688 first time or reactivated donors were tested for iron status and red blood cell count, including CHr. The ferritin index as a marker to indicate absent iron stores (AIS) was calculated. RESULTS: Depending on the number of donations during the preceding 12 months, AIS were detected in up to 21.4% of male and 27.8% of female donors, respectively. IH was present in up to 6.4% male and 16.7% female donors with 2 and 4 preceding donations, respectively. The defined CHr cut-off value was 28.0 pg to detect IH in frequent whole blood donors with AIS, leading to a test specificity of 98.2% (positive predictive value, PPV: 57.7%) in male and of 97.8% (PPV: 82.9%) in female donors. CONCLUSION: Determination of CHr is feasible to detect FID resulting in IH in frequent blood donors. It may help to prevent the development of anemia in frequent blood donors and also can help to decide whether donor deferral or even iron substitution need to be recommended.

What are a patient's current chances of finding a matched unrelated donor? Twenty years' central search experience in a small country.

Between 1988 and 2007, international searches for matched unrelated donors (MUDs) were performed for 1586 Austrian patients. Between 2004 and 2007, a MUD was identified for 76.7% of the patients. Between 1996 and 2003, a donor was identified for 71.3% of the patients, and between 1988 and 1995, only for 53.4% of the patients. Search times of successful searches decreased from 7.7 months in the first period to 1.7 months in the period from 2004 to 2007. However, transplants were not performed in all cases in which a donor was found: only in 61.6% of the patients between 2004 and 2007, in 53.4% between 1996 and 2003 and in 29.6% between 1988 and 1995. Multivariate analysis determined that having a common HLA type was the most important variable impacting on finding a MUD for a patient. Factors that most strongly influence a patient's access to transplant were the patient's European origin and a short time between diagnosis and start of donor search. The strongest factor for both finding a donor and being transplanted was a search being performed during more recent years: patients' chances increased from year to year.

Function and activation state of platelets in vitro depend on apheresis modality.

BACKGROUND AND OBJECTIVES: In multicomponent collection, various blood components are prepared during one apheresis process. The aim of this prospective crossover study was to compare the function, metabolic parameters and activation state of fresh and stored platelets (PLTs) collected by two different cell separators. MATERIALS AND METHODS: Twenty-four donors underwent apheresis on each of two cell separators (Fenwal Amicus(®) and CaridianBCT Trima Accel(®)) with an interval of at least 2 months between donations. Per donation, one double dose of PLT concentrate (PC) and one unit of packed red-blood-cells were collected. In total, 48 single unit PCs were tested for pH, glucose, bicarbonate, lactate, potassium and LDH concentration during 7 days of storage. PLT function was analysed by aggregometry, rotation thrombelastometry and hypotonic shock response. The PLT surface expression of P-selectin (CD62P) and LAMP-3 (CD63) was estimated by flow cytometry. RESULTS: During storage, metabolic parameters were well maintained in both groups, but levels of glucose and pH were significantly lower, while lactate and LDH were significantly higher in Amicus(®)-PCs. Amicus(®)-derived PLTs were significantly more activated as evidenced by higher CD62P and CD63 expression. In parallel, the in vitro function of Amicus(®)-PLTs was significantly reduced compared to Trima(®)-PLTs. CONCLUSION: In multicomponent apheresis, standardized PLT collection is effective and well tolerated. The higher activation of Amicus(®)-derived PLTs may be because of the divergent centrifugation modalities during collection. Possible consequences for the clinical outcome of thrombocytopenic patients will be evaluated in further trials.

Severe thrombocytopenia due to host-derived anti-HPA-1a after non-myeloablative allogeneic haematopoietic stem cell transplantation for multiple myeloma: a case report.

BACKGROUND AND OBJECTIVES: Host- or donor-derived alloimmune thrombocytopenia can develop after non-myeloablative allogeneic haematopoietic stem cell transplantation (HSCT). We report the first case of host-derived HPA-1a antibodies. CASE REPORT: A 52-year-old male patient received HSCT from his human leucocyte antigen (HLA)-A, -B, -C, -DR identical brother after reduced intensity conditioning. Bilinear engraftment around day 12 was accompanied by a continuous decrease of platelet counts. We investigated for platelet antibodies because of a progressive decline of platelet counts and refractoriness to platelet transfusions. METHODS: The patient's serum was tested by enzyme-linked immunosorbent assay (ELISA), a solid phase assay and monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay. Recipient's DNA from the time before HSCT and donor's DNA were genotyped for human platelet antigens. RESULTS: Serum obtained on day 15 after HSCT reacted strongly with the donor's platelets due to host-derived anti-HPA-1a- and anti-HLA I antibodies. Serum samples from days 39, 45 and 65 after HSCT contained only anti-HLA I; no antibodies were detectable on day 149. Platelet counts increased on day 20 spontaneously. The decrease of the antibodies accompanied by the increase of the platelet counts suggests progressive elimination of residual host cells. CONCLUSIONS: The HPA-1a antibodies affected thrombopoietic engraftment and the success of platelet transfusions.

Kell expression on myeloid progenitor cells.

Kell is one of the major human red blood cell groups and comprises 22 antigens. These antigens are produced by alleles located on chromosome 7, including sets of antithetical antigens such as Kell (K, K1) and cellano (k, K2), which differ in a single amino acid change (T193M). It consists of a 93-Kd transmembrane glycoprotein that is surface-exposed and shares sequence and structural homology with zinc endopeptidases, which are involved in regulating bioactive peptides. Anti-Kell antibodies have been shown to suppress fetal erythropoiesis. Recently published data indicate a similar effect on myeolopoiesis and megakaryopoiesis. Substantial thrombocytopenia in fetuses affected with HDN due to anti-K antibodies led to the discovery of the inhibitory effect of Kell-related antibodies on CFU-MK growth. In addition to its inhibitory effect on BFU-E growth, anti-Kell antibodies significantly reduced CFU-GM colony formation from haematologically normal individuals. Moreover, anti-cellano and anti-Kp(b) antibodies also inhibited the growth of CFU-GM from antigen positive MNC. These data indicate that Kell is not restricted to erythroid blood cells, but is expressed on a broader spectrum of haematopoietic cells than previously believed.

Kinetics of CFU-Mk after automated plateletpheresis.

BACKGROUND AND OBJECTIVES: Platelet count, thrombopoetin (TPO) level and the compartment of megakaryocyte progenitor cells (CFU-Mk) are major determinants in the regulation of thrombopoiesis. The aim of this study was to investigate the potential changes in the compartment of CFU-Mk and their correlation with serum TPO levels and platelet count after plateletpheresis. MATERIALS AND METHODS: Twelve healthy individuals were randomly assigned to undergo single-donor plateletpheresis. A collagen-based in vitro culture system was used to determine the number of peripheral blood (PB) CFU-Mk before and after donation and on days 1, 4 and 7 thereafter. TPO levels were measured by a specific enzyme-linked immunosorbent assay and whole blood counts were performed using an automated cell counter. RESULTS: The pre-apheresis platelet count (mean +/- SEM: 276 +/- 13 x 10(9)/l) decreased after plateletpheresis to a nadir of 194 +/- 8 x 10(9)/l (P < 0.001), showed a gradual increase on days 1 and 4, and reached pre-apheresis values by day 7 (280 +/- 11). The serum TPO levels were found to be significantly increased on days 1 and 7 as compared to baseline levels (baseline value 103.3 +/- 18.5 pg/ml versus day-1 value 135.8 +/- 25.8 pg/ml and day-7 value 132 +/- 30.19 pg/ml; P < 0.03 and P < 0.03, respectively). The numbers of CFU-Mk in PB were significantly elevated on day 4 only (125 +/- 21 colonies/ml of PB versus pre-apheresis values of 68 +/- 18 colonies/ml of PB, P < 0.005). CONCLUSIONS: These findings suggest that platelet loss during plateletpheresis affects thrombopoiesis at the progenitor cell level, probably through alterations in TPO plasma concentrations.

IL-10 increases the number of CFU-GM generated by ex vivo expansion of unmanipulated human MNCs and selected CD34+ cells.

BACKGROUND: Ex vivo expansion strategies with different cytokine combinations are currently used by several groups as a means of increasing the number of HPCs for a variety of special clinical applications. Because there is little information on the potential role of IL-10 in such ex vivo expansion models, the effect of this cytokine on the generation of myeloid progenitor cells in suspension cultures was investigated. STUDY DESIGN AND METHODS: On the basis of data from the literature and from new experiments, the combination of SCF and IL-3 at concentrations of 100 ng per mL and 100 U per mL, respectively, was chosen as the standard cocktail. The addition of IL-10 to such cultures resulted in a marked and dose-dependent potentiation of myeloid progenitor cell production. RESULTS: Using unmanipulated leukapheresis components from 13 individuals (including lymphoma and cancer patients and normal donors), the expansion multiple of CFU-GM after 14 days as compared with pre-expansion values was 9.54 +/- 2.31 times by SCF/IL-3 and 46.38 +/- 7.37 times by the combination of SCF/IL-3 and 100 ng per mL of IL-10 (p<0.001). IL-10 also potentiated CFU-GM generation from selected CD34 PBMNCs (n = 9) with an expansion of 17.22 +/- 7.04 times versus 45.67 +/- 16.78 times using the SCF/IL-3 and SCF/IL-3/IL-10 combination, respectively (p<0.05). Moreover, expansion-promoting effects of IL-10 were observed in liquid cultures containing MNCs from bone marrow (n = 4) and cord blood (n = 3), but did not reach statistical significance because of the small number of samples. CONCLUSION: These results suggest IL-10 as a useful cytokine to optimize progenitor cell-expansion strategies for clinical application.

Kell is not restricted to the erythropoietic lineage but is also expressed on myeloid progenitor cells.

Anti-Kell antibodies have been shown to suppress fetal erythropoiesis, but little is known about their effect on myelopoiesis. We analysed the effect of Kell-related antibodies on granulocyte-macrophage colony-forming units (CFU-GM) growth in semisolid medium using peripheral blood mononuclear cells (PBMNCs) from haematologically normal individuals. In addition to its inhibitory effect on erythroid burst-forming units (BFU-E) growth, anti-Kell antibodies significantly reduced CFU-GM colony formation from Kell-positive individuals but not from Kell-negative donors. Moreover, anti-cellano and anti-Kpb antibodies also inhibited the growth of CFU-GM from antigen-positive MNCs. These data indicate that Kell is not restricted to erythroid blood cells, but is also expressed on myeloid progenitor cells.

The determination of metabolite M17 and its meaning for immunosuppressive cyclosporin therapy.

Cyclosporin A (CyA) is intensively metabolized by the hepatic cytochrome p450 III monooxygenase A system in the human liver, the most important metabolites being M1, M17, and M21. Because CyA and its metabolites have nephrotoxic, hepatotoxic, and neurotoxic side effects, CyA dosage must be calculated to avoid the risk of organ rejection through underdosage and toxic organ damage through overdosage or accumulation of metabolites. In this study, we determined the whole-blood concentrations of cyclosporin and metabolite M17 by high-pressure liquid chromatography (HPLC) and by monoclonal specific and polyclonal nonspecific fluorescence polarization immunoassay (Abbott) in patients after immunosuppressive treatment. Patients with different resorption and metabolization rates showed high individual variations. CyA concentrations in patients with good liver function and low concentrations of CyA metabolites showed a good correlation between the HPLC and the FPIA (TDx-monoclonal assay) methods in ranges between 25 and 180 ng/mL. TDx-monoclonal was not always as precise as HPLC. In cases of metabolic disorders, we found false high CyA concentrations assayed with the immunologic method, caused by a crossreaction of the elevated metabolite concentration. We found that HPLC rendered more information about the extent of immunosuppressive activity and the metabolization rate and showed a good correlation with the concentration of metabolite M17 and total metabolites measured with the Abbott CyA polyclonal kit.

[Computer-assisted evaluation of a "medium resolution" HLA-DRB oligotyping of bone marrow donors]. / Computerunterstützte Auswertung einer "Medium resolution"-HLA-DRB-Oligotypisierung von Knochenmarkspendern.

One drawback of a 'medium resolution' generic dotblot oligonucleotide typing of the HLA-DRB locus has been the time consuming evaluation of the collected SSO-hybridisation datas. We developed a program on the basis of MS-ACCESS, written in visual basic, called HIDE (HLA Intelligent Data Evaluation) to manage this problem. HIDE offers a complete data management for the donor and the incoming sample, supports a membrane based data entry of the hybridisation-reactions and multiple testing of a sample. If an evaluation shows more than one matching allele combination, the result is summarized according to the main DRB-groups. The flexible structure of the program allows the entry of additional allelspecific reaction-patterns as well as the adaption to different SSO-typingsets.

[Walkaway systems in general practice: Behring ELISA Processor III]. / Walkaway-Systeme in der Praxis: Behring Elisa Processor III.

The evaluation of a new Walkaway instrument (Behring Elisa Processor III) for testing HBsAG, anti-HCV and anti-HIV 1/2 in three blood transfusion services with different sample volumes (from 60,000 to 450,000/year) gave the following results: the instrument fulfills the promises of being a real Walkaway system under routine conditions, saving labor and gaining security. The instrument must work in agreement with GLP rules, and all test steps are documented. The organization of the laboratory becomes more transparent and less flexible.

Congenital sideroblastic anemia successfully treated by allogeneic bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) was carried out on a 34-month-old boy with congenital sideroblastic anemia. The patient had been red blood cell transfusion dependent since the age of 7 weeks. He did not respond to therapy with pyridoxine and developed secondary progressive hemosiderosis. The preparatory regimen consisted of busulfan (3.5 mg/kg for 4 days) and cyclophosphamide (50 mg/kg for 4 days). Full engraftment of donor bone marrow was achieved and effective hemopoiesis is still maintained 3 years after BMT.

Intrathyroidal dendritic cells, epitheloid cells, and giant cells in iodine deficient goiter.

Immunohistochemistry and immunofluorescence were performed on thyroid sections of 44 consecutive patients undergoing thyroid surgery for goiter due to iodine deficiency. Sections were compared with specimens from ten individuals without goiters from the same endemic area, with specimens from ten sporadic nontoxic goiter patients, and with specimens from an area with sufficient iodine supply from nine healthy subjects. Cells were characterized using monoclonal antibodies to the CR3 receptor (CD11b) and the p150/95 antigen (CD11c) present on macrophages, to HLA-DR, to antigen presenting cells (RFD1), to T helper (CD4) and to T suppressor/cytotoxic cells (CD8), and with a polyclonal antibody to human cytokeratin. In iodine deficient goiters, focal aggregates were found of RFD1-positive dendritic cells. Furthermore, RFD1-positive epitheloid cells were seen. In 27% of cases, these epitheloid cells completely filled the thyroid follicles. Within the epitheloid cell clusters, multinucleated giant cells could be detected that carried the macrophage markers. Dendritic cells, epitheloid cells, and giant cells were strongly HLA-DR positive. In nongoitrous thyroids from the endemic area such aggregates could also be seen but they were more sparse and were RFD1 negative. Giant cells were absent there. In normal thyroids with sufficient iodine supply, only a few isolated dendritic cells were seen. All except RFD1, which was negative, showed the same marker pattern. In sporadic nontoxic goiters from an area with sufficient iodine supply, dendritic cells occurred in much higher numbers than in the normal thyroids from that area, and they were RFD1 positive. They never aggregated as in iodine deficiency, and giant cells were not observed. These observations on iodine deficient goiter strongly suggest involvement of active antigen-presenting cells in this disorder. However, the immunohistologic difference between this disease and sporadic goiter suggests different underlying mechanisms.

[Clinical application of alkaline phosphatase bone-isoenzyme determination with acetate foil]. / Die klinische Einsatzmöglichkeit der Alkalische-Phosphatase-Knochenisoenzymbestimmung mittels Azetatfolie.

Clinical application of alkaline phosphatase bone isoenzyme determination using acetate foil is investigated in patients with known underlying disease and is compared with diagnostic evaluation of 112 bone-scans. The separation of alkaline phosphatase isoenzymes proves a valuable diagnostic support of bone scintigraphy. Isoenzyme determination moreover reveals primary and secondary bone changes in early stages of the disease and also offers the possibility of quantitative follow-ups.

[Primary biliary liver cirrhosis]. / Die primär biliäre Leberzirrhose.

Primary biliary cirrhosis, or chronic destructive nonsuppurative cholangitis, is a condition of chronic cholestasis, in which small intrahepatic bile ducts in the portal zones of the liver become progressively destroyed. The etiology of primary biliary cirrhosis is unknown, but the observation of (a) mitochondrial antibody, (b) elevated serum levels of IgM and (c) circulating immune complexes and (d) impaired lymphocyte transformation (- cooperation) strongly suggest, that disordered immune responses play a major role in the initiation or progression of this chronic hepatic lesion.

Defects of leukocyte locomotion in multiple myeloma.

23 patients with multiple myeloma of IgG, IgA and light chain type, one patient with benign monoclonal IgE gammopathy and 23 healthy controls were investigated for their leukocyte locomotion (LL), for the ability of their sera to generate chemotactic factors upon activation with zymosan, and for inhibitory effects of their sera upon LL. While no significant impairment of the patients' LL was observed when casein was used as cytotaxin, LL was markedly reduced in myeloma patients with zymosan activated control serum as chemoattractant. The ability of serum to generate chemotactic factors upon activation with zymosan was similar in patients and controls, but preincubation of control leukocytes with sera derived from patients with multiple myeloma caused a significant inhibition of LL (p less than 0.001). This LL-inhibitory serum effect was not correlated with the type of paraproteinemia. We conclude that the impairment of LL in multiple myeloma and the LL-inhibitory serum effect might contribute to the increased susceptibility to infections in that disorder.

[Diagnostic problems in monoclonal gammopathies]. / Diagnostische Probleme bei monoklonalen Gammopathien.

This is a report on 123 patients with monoclonal gammopathies at Graz University Medical School. Approximately 75 percent of them were malignant, the rest benign or unclassified. In view of the considerable interest for differential diagnosis benign versus malignant, the criteria for both are given.