Pseudocontamination of blood components with Burkholderia cepacia during quality controls.

We report on a pseudooutbreak of Burkholderia cepacia because of the use of a contaminated disinfectant during quality controls in a university blood bank. No septic reactions associated with transfusions had been reported in patients over the last 6 months. Analysis of the individual quality control procedures showed that a disinfectant based on a quaternary ammonium compound (QAC) had been used in order to disinfect the rubber stopper of the blood culture bottle. B. cepacia was found in a sample taken from this disinfectant, which was prepared with concentrate and tap water according to the manufacturer's instructions. The four isolates (one in disinfectant and three in blood components) were found to be identical in their biochemical reactions and resistance patterns. QAC-based disinfectants are not efficacious against a part of the spectrum of gram-negatives and are therefore inadequate. After introduction of an alcohol-based preparation, no more cases of B. cepacia contamination have been identified.

Alternative pathway activation of the complement system in preterm infants with early onset infection.

The increased incidence of infection in preterm neonates has been related in part to their relative deficiency of most complement components, because complement is known to participate in the defense against bacterial and viral infections. In a prospective study, complement activation products were determined in 52 preterm infants. Twenty preterm infants suffered from proven early onset infection, 11 infants were presumed to suffer from infection, which could not be confirmed. Twenty-one preterm infants without infection or perinatal asphyxia formed the control group. EDTA plasma was obtained within the first 6 h after birth, and follow-up examinations were done in 15 patients with proven infection during the next 24 h. The complement activation products C3a-desArg, C3bBbP, and sC5b-9 were measured with enzyme immunoassay systems. In preterm neonates with early onset infection, a significant elevation of C3a-desArg was found in the very early course of the disease. C3a-desArg generation resulted from alternative pathway activation as shown by a concurrent increase of C3bBbP concentration. In addition, significantly higher concentrations of sC5b-9 predicted infection in the first few hours after birth. Thus, despite very low levels of native complement proteins, preterm babies are able to generate remarkable amounts of activation products of the complement cascade. The elevation of these activation products preceded by hours significant changes of routine laboratory markers of infection, such as leukocyte count, differential blood count, and C-reactive protein. Thus they might help to identify preterm neonates with severe systemic infection earlier than other laboratory parameters.

CMV-induced anti-Sia-b1 cold agglutinin in an immunocompromised patient.

In postinfection cold agglutination, certain cold agglutinin (CA) specificities are associated with distinct infectious agents. The combined occurrence of anti-I and anti-Sia-b1 CAs following Mycoplasma pneumoniae infection has been reported recently. After renal transplantation and hyperacute graft rejection, transiently occurring CAs were observed in an 18-year-old boy. The CAs were characterized by serum cold absorption with sialidase-treated red cells and warm elution from the cells. An anti-Sia-b1 CA could be differentiated from an accompanying low-liter anti-I. Fresh infections with Mycoplasma pneumoniae, Epstein-Barr virus, rubella, and varicella viruses were excluded, but CMV infection was demonstrated. This is the first case of a postinfection anti-Sia-b1 CA associated with CMV infection.

[Rheologic changes in stored erythrocyte concentrates]. / Rheologische Veränderungen gelagerter Erythrozytenkonzentrate.

We studied the effect of two different preservation solutions on mean corpuscular volume (MCV), red cell deformability and flow in narrow tubes in red blood cell concentrates. Blood from 10 healthy blood donors was processed in parallel in SAG-M (S-RBC) as well as in PAGGS-M (P-RBC) in identical aliquots. Samples were studied at days 0, 7, 14, 28 and 42 of storage. MCV was determined using a Du Pont cell counter. Whole cell deformability was determined in a Myrenne Rheodyn. Viscosity reduction in narrow tubes was determined by means of capillary viscosimetry. P-RBC showed a constant MCV over the entire storage period. In contrast, MCV of S-RBC increased and MCHC decreased during storage. P-RBC showed similar deformability and viscosity reduction during storage, whereas deformability decreased and viscosity reduction became less pronounced for S-RBC. Our study shows superior rheological properties of P-RBC. Thus, PAGGS-M may provide better hemoglobin flux and oxygen transport to tissues than SAG-M.

Inflammatory mediators in relation to the development of multiple organ failure in patients after severe blunt trauma.

OBJECTIVE: To evaluate the posttraumatic course of several inflammatory mediators or markers (complement components C3, C3a, terminal complement complex, thromboxane B2, C-reactive protein, elastase, and neopterin) in relation to the development of multiple organ failure and mortality. DESIGN: Prospective study of a selected patient group. SETTING: Surgical intensive care units in three European trauma hospitals. PATIENTS: Patients (n = 56) with severe blunt trauma (Injury Severity Score of > or = 33). INTERVENTIONS: Arterial blood samples were sequentially obtained. MEASUREMENTS AND MAIN RESULTS: Nonsurvivors (n = 8) had significantly higher circulating C3a and elastase concentrations on the first postinjury day, compared with survivors (n = 48). No differences between these groups were found for terminal complement complex, thromboxane B2, C-reactive protein, and the neopterin/creatinine ratio. Five patients died before day 5. Eighteen patients developed multiple organ failure, which was diagnosed from day 5 onward, leaving 33 patients without multiple organ failure. The patients with subsequent multiple organ failure showed significantly higher mean circulating concentrations of C3a (914 +/- 190 [SEM] ng/mL), terminal complement complex (57 +/- 17 U/mL), and thromboxane B2 (275 +/- 37 pg/mL) at the first postinjury day than the patients without multiple organ failure (566 +/- 110 ng/mL, 27 +/- 2 U/mL, and 169 +/- 14 pg/mL, respectively). In patients with multiple organ failure, elastase concentrations were significantly higher on days 2, 3, 4, and 5 postinjury. Neopterin/creatinine ratios, on the other hand, were significantly higher in patients with multiple organ failure when the multiple organ failure had already become established (on days 8 and 10). CONCLUSION: In multiple trauma patients, excessive triggering of the inflammatory cascade-as expressed by complement activation and stimulation of neutrophils producing elastase--plays an important and early role in the development of multiple organ failure.

Red cell destruction in cold agglutinin disease.

OBJECTIVE: The severity of autoimmune hemolytic anemia (AIHA) caused by cold agglutinins (CAs) is known to differ markedly in chronic CA disease as well as in postinfection cold agglutination. The various cold agglutinin specificities and mechanisms of red cell destruction are described. DATA SOURCES: Original papers and reviews of the German and English literature (Medline research); own results. SELECTION CRITERIA: Original papers and reviews of the recent years on studies of the biochemistry and specificity of CAs and the pathophysiology of AIHA. RESULTS: A crucial point for the severity of AIHA caused by CAs is the CA-binding capacity to red cells in vivo. This is reflected by the serologic behavior of the actual CA in vitro, represented by its thermal amplitude. Because most CAs are IgM molecules, red cell destruction by CAs is limited to mechanisms initiated by complement (C) activation. In recent years, several CA specificities, in addition to anti-I/i, have been identified on a serological and biochemical basis. CAs of the IgG and rarely of the IgA isotypes have been found. CONCLUSION: The CA-induced red cell destruction does not only depend on CA titer or its thermal amplitude. The severity of CA-induced AIHA may also depend on CA isotype and/or specificity. Therefore, the complement activation capacity of CAs with a given isotype but different specificities has to be elucidated in further studies.

Plasmapheresis in severe septic shock with disseminated intravascular coagulation.

An 18-year-old female with CNS relapse of acute lymphoblastic leukemia after previous complete remission of the disease underwent chemotherapy. Due to the therapy she suffered from profound suppression of bone marrow with consecutive thrombocytopenia and leukopenia. Despite prophylactic treatment, severe septicemia occurred with septic shock, hemolysis and disseminated intravascular coagulation (DIC). As the clinical course became uncontrollable by means of conventional therapy, including broad-spectrum antibiotics, substitution of fresh frozen plasma, antithrombin III and heparin therapy, plasma exchange was used as a rescue therapy. This method succeeded in effective replacement of clotting factors and normalization of coagulation, in removal of fibrinogen degradation products and probably of toxins and shock mediators. The patient recovered from shock.

Complement activation in newborn infants with early onset infection.

The complement system is an important element in host defense. Quantitative deficiencies of total hemolytic complement activity and decreased C3 levels were reported in sera from normal neonates. However, little is known about complement activation products in the newborn. In a prospective study, complement activation products were determined in 32 healthy term neonates, in 41 neonates with colonization of their mothers, in 15 colonized neonates, and in 10 neonates with early onset infection. In all newborns, EDTA plasma was obtained within the first 6 h of life. The anaphylatoxin C3a-desArg was determined with a novel ELISA using an MAb reacting with a neoepitope of C3a-desArg. C3bBbP (alternative pathway convertase) and C1rsC1-inactivator (activation product of classical pathway) were measured with double-sandwich ELISA. C3 was determined by radial immunodiffusion. Plasma concentrations of C3a-desArg were similar in healthy term neonates and healthy adults, whereas diminished C3 levels were observed in the newborn infants. There were no significant differences between healthy neonates, neonates with colonized mothers, and colonized neonates. In neonates with infection, a significant elevation of C3a-desArg was found at the onset of the disease, resulting from alternative pathway activation. In contrast, the C1rsC1-inactivator complex showed no significant differences among healthy, colonized, and infected neonates. The anaphylatoxin C3a mediates inflammatory reactions such as vasodilatation and an increase in microvascular permeability and might therefore play an important role in severe neonatal infection.

Interleukin-4 augments production of the third complement component by the alveolar epithelial cell line A549.

The elements of allergic inflammation and the involvement of helper T lymphocytes are increasingly being recognized in the immunopathogenesis of asthma. Allergen exposure leading to the activation of allergen-specific T cells present in the lung can result in the release of cytokines which in turn can locally stimulate the cellular constituents of the lung. The airway epithelial cells may be the key participants in such an interaction. Therefore, we examined the ability of T-cell-derived IL-4 to modulate the production of C3 and C5 by the human type-II pneumocyte cell line A549, which is known to produce all the components and the regulatory proteins of the complement system. For estimation of C3 an ELISA detecting native C3 was used. Following stimulation of A549 with hrIL-4 a dose-dependent (1-50 U/ml) enhancement of C3 production was observed, which reached its maximum (5-fold of unstimulated cells) at 48 h and gradually declined thereafter. Concentrations of hrIL-4 higher than 50 U/ml did not further increase C3 production. In parallel experiments hrIFN-gamma at concentrations between 10 and 50 U/ml stimulated the C3 production to more than twice the quiescent state level within 24 h. In the pneumocyte cell line A549 we demonstrated the expression of a gene for the IL-4 receptor which appears to mediate the biological effect of this lymphokine. A diminution in the functionally active C5, estimated by ELISA at the same time, was observed in supernatants of A549 cultures following stimulation with hrIL-4 as well as with hrIFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of anaphylatoxin C3a in plasma and bronchoalveolar lavage fluid in trauma patients at risk for the adult respiratory distress syndrome.

OBJECTIVE: To determine the generation of anaphylatoxin C3a in plasma and bronchoalveolar lavage fluid in trauma patients at risk for the adult respiratory distress syndrome (ARDS). DESIGN: Prospective study. SETTING: ICU in a university hospital. PATIENTS: Severely traumatized patients at risk for the ARDS (n = 25). INTERVENTION: EDTA plasma samples and bronchoalveolar lavage fluid were obtained. MEASUREMENTS AND MAIN RESULTS: Complement proteins C3, C4, C5, and the inhibitors C1-inhibitor, Factor H, and Factor I were quantitated in EDTA-plasma samples obtained every 6 hrs during the first 48 hrs after ICU admission and every morning from days 4 to 14 after injury. In bronchoalveolar lavage fluid, the complement activation production of C3a-desArg was quantitated and the volume of epithelial lining fluid was calculated. All patients showed a decrease of the complement proteins C3, C4, C5 and of the inhibitors C1-inhibitor, Factor H, and Factor I during the first 24 hrs, indicating complement consumption. Patients developing ARDS (n = 11) showed significantly higher C3 concentrations and a higher C3a/C3 ratio in the first few hours after multitrauma. Follow-up bronchoalveolar lavages demonstrated highly increased amounts of C3a in epithelial lining fluid during the first 24 hrs, mainly in ARDS patients and, to a lesser degree, in non-ARDS patients. To determine the origin of C3a in bronchoalveolar lavages, the ratio of C3a in epithelial lining fluid and plasma was calculated. CONCLUSION: The C3a of epithelial lining fluid to plasma ratio was extremely high in patients developing ARDS, but even the non-ARDS group had a ratio greater than 1, indicating that a substantial local complement activation occurs in the lung.

[The critical hemoglobin value in newborn infants, infants and children]. / Kritische Hämoglobinwerte bei Neugeborenen, Säuglingen und Kindern.

The optimum and critical hemoglobin concentrations are determined by the oxygen demand of the tissues and several oxygen transport parameters (i.e., blood flow, arterial oxygen saturation, oxygen affinity of hemoglobin, and the critical venous oxygen pressure). Most of the oxygen transport parameters change markedly during the first weeks after birth. Oxygen consumption and cardiac output in neonates are three times those of adults on a body weight basis. Due to the high oxygen affinity of fetal hemoglobin, the oxygen unloading capacity of hemoglobin in neonates is about 50% less than in adults. From oxygen transport parameters and oxygen consumption we have calculated the optimum and the critical hemoglobin concentrations for preterm and full-term neonates during the first weeks after birth. A hemoglobin concentration of 15 g/dl appears optimal for preterm and full-term infants at birth as well as for adults. The calculated minimum acceptable hemoglobin concentration is 6 g/dl for children and adults, 12 g/dl for preterm infants and 11 g/dl for full-term neonates at birth. Due to the postnatal decrease in oxygen affinity, the minimum acceptable hemoglobin concentration decreases by approximately 1 g/dl/week for the first 5-6 weeks until the minimum value of 6 g/dl for children and adults is reached. The minimum hemoglobin concentration should be 2 g/dl higher in patients who require increased oxygen or suffer from other serious disorders. A minimum hemoglobin concentration of 10 g/dl is recommended in children with leukemia or other oncological disease. In infants and children with chronic hypoxemia (cyanotic congenital heart disease) the minimum hemoglobin concentration should be increased by the percentage of arterial oxygen desaturation.

Complement activation and the prognostic value of C3a in patients at risk of adult respiratory distress syndrome.

In vivo and in vitro studies have shown that complement activation plays an important role in the pathogenesis of the adult respiratory distress syndrome (ARDS). In a prospective study of polytrauma patients at risk of ARDS (n = 38) complement parameters were determined over a period of 14 days in serial plasma samples (obtained every 6 h during the first 48 h). Polytrauma induced a rapid and remarkable complement activation. Low levels of the complement proteins C3, C4, C1 inhibitor (C1 INH) factor I and factor H during the first 48 h indicated complement consumption in all patients. Elevated C3a levels in the first few hours after injury were associated with the later development of ARDS. A more sensitive indicator than C3a alone was the calculated C3a:C3 ratio discriminating ARDS and non-ARDS patients. A second rise of C3a levels and C3a:C3 ratio from day 4 on paralleled the course of extravascular lung water. To assess the mode of complement activation, the activation-specific protein complexes C1rC1s-C1 INH and C3b(Bb)P were measured in some of the patients. We demonstrate that in the first 48 h complement activation occurred via the alternative pathway only and was later followed by an additional activation via the classical pathway. Our observations suggest that monitoring of C3a and C3 in plasma can identify polytrauma patients at high risk for ARDS at an early stage of the disease.

Quantitation of the anaphylatoxin C3a in the presence of C3 by a novel sandwich ELISA using monoclonal antibody to a C3a neoepitope.

C3a levels in plasma are usually measured by a competitive inhibition radioimmunoassay (RIA) using 125I-labelled C3a-desArg and antibodies to C3a capable of detecting C3a determinants which are also present on the native C3. Therefore, prior to the assay native, non-cleaved C3 has to be removed completely from the C3a-containing sample by precipitation. We developed a new rapid two-site sandwich ELISA system for the quantitation of C3a-desArg in plasma. This immunoassay uses a monoclonal antibody (mAb H466) reacting with C3a-desArg but not with C3. The reactivity of mAb H466 with a neoantigenic determinant of C3a-desArg permitted the direct quantitation of C3a-desArg without removal of C3 from the sample. The mAb H466 was used as a capture antibody and bound C3a-desArg was detected with a second peroxidase-labelled anti-C3a mAb. The lower limit of detection of C3a-desArg in this ELISA was 1 ng/ml. The C3a-desArg levels measured in the plasma samples of various patients were found to differ over a wide range. A good correlation was observed between the results obtained in the RIA and those obtained in the ELISA (r = 0.95). High levels of C3a-desArg were detected in plasma from patients with multiple trauma and patients undergoing haemodialysis. The C3a-desArg assay described should facilitate the routine quantitation of C3a in samples of plasma.

The C terminus of the anaphylatoxin C3a generated upon complement activation represents a neoantigenic determinant with diagnostic potential.

Activation of the C component C3 results in generation of the anaphylatoxin C3a. The C3a polypeptide chain consists of 77 amino acids. The active site of this potent mediator, which also has immunoregulatory function resides in its C terminus. This report demonstrates that the C terminus of C3a (C3a-desArg) exposed by proteolytic cleavage from C3 represents a neoantigenic determinant. Two mAb specific for this epitope were obtained after immunization with the synthetic octapeptide (OP) Arg-Ala-Ser-His-Leu-Gly-Leu-Ala [C3a(69-76)] coupled to the carrier keyhole limpet hemocyanin (KLH). These anti-C3a(69-76) antibodies (H453 and H454) reacted in an ELISA system with C3a and KLH-OP but not with C3 or with KLH alone. Free OP efficiently blocked binding of the antibodies to C3a, whereas binding of another anti-C3a mAb (H13) remained unaffected. In immunoblotting analysis, the anti-C3a(69-76) mAb reacted with purified C3a but failed to react with the denatured, noncleaved C3. A novel quantitative C3a-ELISA was established with the anti-C3a(69-76) mAb. It had a sensitivity in the nanogram range (1 to 5 ng/ml). The C3a determination was not impaired by the presence of high concentrations of C3. Therefore, C3 removal was not required in contrast to the previously described C3a assays. This C3a ELISA might facilitate clinical C3a quantitation, e.g., in samples from patients with adult respiratory distress syndrome. In these patients, C3a determination in the early phase of the disease is of diagnostic relevance and has prognostic value.

Functional analysis and quantification of the complement C3 derived anaphylatoxin C3a with a monoclonal antibody.

The C3 fragment C3a belongs to the anaphylatoxins. It has immune regulatory activity and contributes to the pathogenesis of the adult respiratory distress syndrome (ARDS). The low molecular weight (9 kD) of C3a complicates the production of antibodies to C3a. We obtained a monoclonal antibody (designated H13) to human C3a. It reacts with C3a or C3a-desArg and with native C3 but not with C5 or C5a. In immunoblot analysis it reacts with the alpha- but not with beta-chain of C3 and binds to a protein with a mol. wt of about 10 kD present in zymosan-activated sera which is only marginally detectable in nonactivated serum and absent in plasma. H13 crossreacts with the analogous proteins of rabbit, guinea pig and sheep. H13 has the capacity to bind 125I-radiolabelled C3a efficiently but fails totally to react with 125I-C5a or with other C3 alpha-chain fragments. H13 blocks C3a functional activity. It markedly inhibits C3a-induced 3H-serotonin release from platelets in vitro and similarly inhibits the C3a-induced extravasation of Evans blue into the skin in vivo. H13 does not interfere with the haemolytic activity of C3. An ELISA system was established using H13 which permits quantification of C3a in sera of polytrauma patients. The antibody H13 should facilitate further functional analysis of C3a in experimental systems. It should be useful for quantification of C3a in diagnostic assays and also for application in immunopathology.