Impact of constant storage temperatures and multiple warming cycles on the quality of stored red blood cells.

BACKGROUND: Red blood cells (RBCs) are routinely stored in liquid state at temperatures below 6°C, and RBC unit core temperature should not exceed 10°C during transport. Since the critical temperature of 10°C was chosen mostly arbitrarily, this study investigated the effect of both constant temperature settings as well as multiple rewarming cycles on stored RBCs with respect to morphology, biochemical parameters and haemolysis. MATERIALS AND METHODS: Buffy coat-depleted filtered RBCs were used as standard products. RBCs were stored at 1-6°C (reference group, n = 12), 13 and 22°C (test groups, n = 12 each) or stored at 1-6°C and warmed up five times to 10, 13, or 22°C for a period of 24 h each. Various biochemical parameters were measured weekly. RBCs were further investigated using electron microscopy. RESULTS: Red blood cells stored constantly at 13 or 22°C showed stable haemolysis rates until day 28 and day 14, respectively. RBCs stored at 1-6°C with five warming-up periods to 10, 13 or 22°C each lasting 24 h (total 120 h) did not exceed the limit of the haemolysis rate at the end of storage. Differently shaped erythrocytes were found in all samples, but more crenate erythrocytes appeared after 42 days of storage independent of temperature profiles. CONCLUSION: Red cells can be kept at constant temperatures above 6°C without apparent harmful effects at least until day 14, whereas multiple warming cycles for no longer than 24 h at 10, 13 or 22°C with subsequent cooling do not cause quality loss as assessed using the in vitro assays employed in this study.

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.

Detection of a new HLA-A allele, designated HLA-A*32:53.

The novel allele A*32:53 differs from A*32:01 by a single nucleotide substitution in exon 4 (position 649G>A).

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.

Core and surface temperatures in a red-blood-cell unit during storage and transport.

BACKGROUND AND OBJECTIVES: Temperature tracing of stored red-blood-cell concentrates (RBCs) is inevitable with respect to RBC quality control. RBC temperature, which should not exceed 10°C, is usually assessed by devices attached to the surface of the RBC pouch, assuming that surface temperature adequately represents the thermal state of RBC. We investigated under which conditions this assumption is true. MATERIALS AND METHODS: Eighteen thermocouples (TC) equidistantly mounted on a two-layer plastic grid were installed in a pouch to determine temperature distribution in the unit. Two TCs were attached to each side of the bag to evaluate surface temperatures. One was further installed in each investigation room to assess ambient room temperatures. Temperature distributions and time-temperature courses were measured under constant temperatures and various warming and cooling conditions. RESULTS: At homogeneous storage temperatures, only small gradients were measured between core and surface temperatures. Removed from cooling chamber to room temperature or back from room to storage temperature, core and surface time-temperature curves drifted apart. Surface and core temperature diverged the more, the faster ambient temperatures altered. The situation could be stabilized by thermal isolation: handled in a transport box, or even better in an air cushion envelope, surface and core courses approached and ultimately closely followed each other, respectively. CONCLUSION: RBC temperature monitoring devices attached to the surface of the RBC pouch very well describe the core temperature under constant temperature conditions. During transport, thermal isolation of the RBC unit is necessary to control RBC temperature precisely by surface temperature measurements.

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.

A new HLA-A*26 variant, A*2637 identified by haplotype-specific extraction and sequencing-based typing.

The novel allele A*2637 differs from the common A*260101 by a single nucleotide substitution in exon 2 (position 186 C>G) causing an amino acid exchange (SER>ARG).

[Blood clots in erythrocyte concentrates during transfusion]. / Blutkoagel in Erythrozytenkonzentraten während der Transfusion.

BACKGROUND: The presence of multiple blood clots in leucocyte-depleted erythrocyte concentrates during a transfusion gave rise to an investigation to find the exact cause. MATERIALS AND METHODS: Determination of the various blood group systems was carried out using the gel centrifugation method and also the polymerase chain reaction (PCR) using sequence-specific primers. In addition the human leucocyte antigens (HLA) class 1 and class 2 markers were determined with molecular biological methods. RESULTS: The erythrocytes in the blood bags containing the blood clots showed a mixed-field agglutination in each blood group where the donor and recipient had different phenotypes. The HLA groups, however, could be solely attributed to the patient, since during the preparation of erythrocyte concentrates all leucocytes are removed and only very few residual cells containing DNA are present. CONCLUSIONS: To the best of our knowledge, this is the first detailed report on blood clots from patient blood in erythrocyte concentrates, which occurred during a transfusion. The retrograde filling of the blood bag with patient blood during the transfusion led to coagulation in the bag. Therefore, careful attention must be taken when dealing with stored blood and corresponding training must be regularly carried out.

Thrombin generation before and after multicomponent blood collection.

The development of apheresis technology has increased efficiency in donor blood use by collecting specific blood components in several combinations. The question of donor safety raised by the contact of donor blood with foreign, only in part biocompatible surfaces remains. The aim of this study was to estimate the effect of multicomponent blood collection on thrombin generation performing an overall function test of coagulation. DONORS, METHODS: 26 blood donors were included. Per apheresis two units of platelets and one unit of RBCs were collected by two cell separators (Amicus and Trima Accel). Each donor underwent the procedure on both apheresis systems. Samples were collected before, immediately after, and 48 hours after apheresis. Thrombin generation was measured by means of calibrated automated thrombography (CAT). RESULTS: CAT-data changed only slightly and no significant changes were seen before, immediately after, and 48 hours after apheresis. The parameters did not differ significantly between the two different apheresis devices. CONCLUSION: No change in parameters of continuous thrombin generation occurred, suggesting that apheresis did not lead to severe alterations in the haemostatic system.

Chromatographically purified immunoglobulin G of endemic and sporadic goiter patients stimulates FRTL5 cell growth in a mitotic arrest assay.

A strain of differentiated rat thyroid cells (FRTL5) in continuous culture was used to study the presence of thyroid growth-promoting immunoglobulins (TGI) in the serum of patients with endemic and sporadic euthyroid goiters. To identify true in vitro cell proliferation a microscopic mitotic arrest assay was used. Immunoglobulins G (IgGs) were prepared with QAE-Sephadex A-50 or protein-A-Sepharose. A positive growth stimulation index was found in IgG preparations of 65 of 71 patients with endemic goiter and in 9 of 14 IgG preparations of patients with sporadic goiter. IgG preparations of 15 control subjects from an area where endemic goiter due to iodine deficiency does not occur and of 18 subjects without iodine deficiency and without thyroid enlargement living in the endemic area did not stimulate FRTL5 cell growth. FRTL5 cell growth stimulation with IgGs of these euthyroid goiter patients could only be detected when IgG was tested in combination with a small dose of TSH. Immunoprecipitation with polyclonal and monoclonal antihuman IgG was able to abolish the growth-promoting effects. In 32 blinded samples the Feulgen cytobiochemical assay, formerly used to detect TGI, was compared with the FRTL5 mitotic arrest assay. The two methods showed similar results. Our observations of chromatographically purified IgG promoting thyroid cell proliferation in vitro provide good evidence that IgG was responsible for thyroid cell growth in vitro and suggest that autoimmune growth mechanisms may be involved in the pathogenesis of both endemic and sporadic goiters.

A rapid immunocytochemical assay for CMV detection in peripheral blood of organ-transplanted patients in clinical practice.

This study describes clinical experience with a rapid method for diagnosis of cytomegalovirus infection in organ-transplanted patients, based on the detection of CMV-specific antigens in peripheral polymorphonuclear cells with a mixture of monoclonal antibodies. This CMV-pp65 assay was formerly called the "CMV immediate early antigen assay." A group of 180 organ-transplanted patients were examined with this assay; 75 of them could be observed from the date of transplantation. These 75 patients consisted of two groups: 59 kidney transplant patients receiving no CMV hyperimmunoglobulin prophylaxis (group I), 13 heart-transplanted patients, and 3 liver transplanted patients receiving prophylaxis (group II). Group III consisted of 105 patients who had been transplanted ca. 2 years before starting this study. In group I, 26 (44%) were CMV-pp65-positive (13 primary and 13 secondary infections). Fifteen of these 26 (58%) positive patients showed clinical symptoms of CMV infection. Eleven of these 15 (73%) were primary infections. Symptomatic patients had significantly more CMV-pp65-positive cells than asymptomatic patients; 12 patients showed a high number of positive cells and 11 of them developed severe CMV illness. Thirty-three patients were CMV-pp-65-negative (22 CMV IgG-sero-positive, 11 CMV IgG-seronegative). None of them had symptoms of CMV infection. In all patients of group I there were 36 periods of graft dysfunction in which CMV infection had to be differentiated from transplant rejection. In 10 out of 36 there was a CMV-pp65-positive test result and subsequent seroconversion. Treatment of viral infection resulted in improvement of clinical problems. In the remaining 26 episodes no CMV-pp65-positive cells were detected: in 17 cases graft dysfunction was caused by rejection, in 9 cases by other complications. In group II, 13 of 16 patients (81%) were positive in the CMV-pp65 assay (6 primary infections, 7 secondary infections). However, none of them showed clinical signs of CMV infection, regardless of the number of positive cells. No CMV-related graft dysfunction was observed. In group III, CMV infections did not play an important role. The experiences described suggest that this test is a valuable tool in early CMV diagnosis and in differentiating CMV-dependent graft dysfunction from other graft dysfunctions. It allows prompt therapeutic intervention.

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.

Acute midbrain syndrome as an adverse reaction to tetanus immunization.

An acute midbrain syndrome III/IV developed twice after tetanus immunization. The occurrence of nearly identical episodes was remarkable, as well as the relatively rapid return to normal consciousness and neurological status after deep coma. Special emphasis is placed upon the chronological relationship of the coma to the immunization and upon immunological tests (tetanus antibody titres) performed to confirm the diagnosis.

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.

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.

[Clinical immunology and immunopathology of glomerulonephritis (author's transl)]. / Kinische Immunologie und Immunpathologie der Glomerulonephritis.

Many problems of clinical immunology with regard to glomerulonephritis become evident only when basic immunopathological principles are taken into consideration. The objective of this paper is to contribute to a deeper understanding of both clinical and immunological problems concerning glomerulonephritis and its prognosis and immunotherapy by a presentation and discussion of immunopathological processes.

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.

A new HLA-B*44 variant, B*4453, identified by haplotype-specific extraction and sequencing-based typing.

We report the identification of a novel HLA B*44 allele, officially named B*4453, found in an Austrian patient and his two sisters.