Bacteria-induced release of white cell--and platelet-derived vascular endothelial growth factor in vitro.

BACKGROUND AND OBJECTIVES: Poor prognosis after resection of primary colorectal cancer may be related to the combination of perioperative blood transfusion and subsequent development of infectious complications. White blood cell--and platelet-derived cancer growth substances, including vascular endothelial growth factor (VEGF), may be involved in this process. Therefore, we studied the in vitro release of VEGF from white blood cells and platelets stimulated by bacterial antigens and supernatants from stored red cell components. MATERIALS AND METHODS: Eight units of whole blood (WB) and eight units of buffy-coat-depleted red cell (SAGM) blood were donated by healthy blood donors. Subsequently, half of every unit was leucocyte depleted by filtration, and all 32 half-units were stored under standard conditions for 35 days. Just after storage, and on days 7, 21 and 35 during storage, aliquots of the supernatants were removed from the units and frozen at -80 degrees C. WB from other healthy donors was stimulated for 2 h with sodium chloride (controls), with Escherichia coli lipopolysaccharide (LPS) alone, or with LPS plus supernatants from the non-filtered or prestorage leucofiltered WB units (diluted 1:10), or from non-filtered or prestorage leucofiltered SAGM blood units (diluted 1:20) stored for 0, 7, 21, or 35 days, respectively. Similar assays were performed using Staphylococcus aureus-derived protein A as a stimulatory antigen. The concentration of VEGF was determined in supernatants from stored blood and in assay supernatants by using enzyme-linked immunosorbent assay (ELISA). RESULTS: The concentration of VEGF increased significantly (P < 0.0001) in a storage time-dependent manner in non-filtered WB and SAGM blood, while the increase was abrogated by prestorage leucofiltration. The supernatant concentration of VEGF was significantly increased in LPS-stimulated (P = 0.002) and in protein A-stimulated (P < 0.0001) assays compared with controls. Addition of supernatants from stored, non-filtered WB or SAGM significantly increased the assay supernatant VEGF concentration storage-time dependently (P = 0.006) in LPS assays. In protein A assays, only supernatants from non-filtered WB significantly increased the assay supernatant VEGF concentration storage-time dependently (P = 0.022). This additional effect by supernatants from stored blood components was not observed with prestorage leucofiltered blood. CONCLUSIONS: Extracellular VEGF may accumulate in non-filtered red cell components, but this can be prevented by prestorage leucocyte depletion using filtration. In addition, bacterial antigens appear to induce release of VEGF from white blood cells and platelets. Addition of supernatants from stored, non-filtered WB or SAGM blood may increase the VEGF levels in a storage time-dependent manner, while prestorage leucofiltration may prevent further increase by supernatants.

Accumulation of the neutrophil-derived protein YKL-40 during storage of various blood components.

OBJECTIVE AND DESIGN: Post transfusion infectious complications associated with allogeneic blood components may depend on storage time and may be related to extracellular accumulation of bioactive substances during storage. YKL-40 is a glycoprotein located in the specific granules of the neutrophils. While exocytosed it may play a role in inflammation and remodelling of the extracellular matrix. We studied the potential accumulation of YKL-40 in blood components during storage. METHODS: Using a RIA method extracellular accumulation of YKL-40 was determined in supernatants from whole blood, plasma-reduced whole blood, buffy-coat-depleted SAGM (saline-adenine-glucose-mannitol) blood, whole blood leukocyte depleted by prestorage filtration, and whole blood leukocyte depleted by bedside filtration. The blood was donated by volunteer, healthy blood donors, and stored under standard blood bank conditions for 35 days. RESULTS: Extracellular accumulation of YKL-40 increased significantly in a time-dependent manner during storage for 35 days of non-filtered whole blood, plasma-reduced whole blood, and SAGM blood, respectively. Prestorage leukocyte depletion of whole blood prevented extracellular YKL-40 accumulation, while YKL-40 accumulation was not reduced by bedside leukocyte depletion. CONCLUSION: YKL-40 appears to accumulate extracellularly in a time-dependent manner in standard erythrocyte components. Prestorage leukocyte depletion by filtration of whole blood may be an effective procedure to prevent extracellular YKL-40 accumulation during storage of erythrocyte components.

Leucocyte and platelet-derived bioactive substances in stored blood: effect of prestorage leucocyte filtration.

Adverse reactions to transfusion of allogeneic blood may depend on content of leucocytes and platelets and on storage-time of the erythrocyte suspensions. Therefore, we studied the efficacy of prestorage leucocyte reduction by filtration on total content and extracellular accumulation of histamine, eosinophil cationic protein (ECP), eosinophil protein X (EPX), myeloperoxidase (MPO), plasminogen activator inhibitor type-1 (PAI-1) and interleukin-6 (IL-6) in samples obtained from 5 units of SAGM blood, 7 units of plasma-reduced whole-blood and 6 units of whole-blood before and after filtration, respectively. In addition, we analysed supernatants from the same units after storage at +4 degrees C for 0, 21 and 35 d, respectively. The filtration was performed at room temperature within 2-4 h after donation. The substances were analysed by ELISA and RIA methods and we also analysed the donor plasma levels of the same bioactive substances. The total content of histamine, ECP, EPX, and MPO were 10-70-fold higher in all unfiltered erythrocyte products compared to donor plasma concentrations, while PAI-1 content was 15-20-fold higher only in plasma-reduced whole-blood and whole-blood. Prestorage leucocyte filtration significantly reduced the total histamine, ECP, EPX, MPO and PAI-1 content to levels similar to donor plasma levels in plasma-reduced whole-blood and whole-blood, while PAI-1 was still low in filtered SAGM blood. In addition, the levels of extracellular bioactive substances at d 0 after donation and filtration were within the range of concentrations in donor plasma, and there was no time-dependent accumulation during storage for 35 d at +4 degrees C. IL-6 was not detected in either plasma or samples obtained from the blood bags. These results suggest prestorage leucocyte filtration to deplete leucocyte contents to levels, which prevent the previously shown time-dependent accumulation of leucocyte derived bioactive substances in various erythrocyte suspensions. In addition, the PAI-1 results suggest leucocyte filters to reduce the obligatory platelet content in whole-blood products.

Mg+ MNS blood group phenotype: further observations.

Red cells carrying the low-frequency MNS antigen Mg reacted with the only example of anti-DANE, an antibody which had previously defined the GP. Dane (Mi.IX) phenotype. Furthermore, Mg+ cells reacted with the original anti-Mur (serum of Mrs. Murrell), but with none of 14 other anti-Mur. Therefore, Mg+ cells carry both DANE antigen and an atypical Mur antigen. Immunoblotting of membranes from Mg+ cells with anti-M, and with eluates prepared from anti-Mg and Mrs. Murrell's serum demonstrated a glycophorin A (GPA) molecule whose mobility was increased by an apparent M(r) of about 3,000 presumably due to the loss of the three O-glycans known to be absent from Mg-active GPA.

Nosocomial epidemic of Serratia marcescens septicemia ascribed to contaminated blood transfusion bags.

Two cases of transfusion-related Serratia marcescens bacteremia prompted extensive epidemiologic investigations in three independent hospitals. Test tubes and plasma from donors whose blood was drawn into bags from a single production batch were cultured. Analysis of the ribotype of S. marcescens isolates was performed. For comparison, a strain from the production plant and eight other, unrelated bacteremia isolates were examined. In addition, a retrospective national survey was carried out. S. marcescens was cultured from 11 (0.73%) of 1515 blood units, and an additional (third) bacteremic patient was identified. The clinical isolates from three patients, the three units of blood transfused, and the plant-derived strain shared a unique ribotype. The incident is interpreted as a sporadic, bacterial contamination of blood bags with the S. marcescens epidemic strain, occurring during the manufacturing or packaging. A similar incident has not previously been reported. Attention is drawn to the possibility of significant contamination during the complex production of multiple-bag blood collection systems. Guidelines for improved registration and handling of transfusion complications in wards are suggested. Manufacturers should be encouraged to provide blood packs with sterile exteriors, in appropriate, single, outer packages.

Molecular analysis of human glycophorin MiIX gene shows a silent segment transfer and untemplated mutation resulting from gene conversion via sequence repeats.

The human glycophorin (HGp) loci that define the red blood cell surface antigens of the MNSs blood group system exhibit considerable allelic variation. Previous studies have identified gene conversion events involving HGpA(alpha) and HGpB(delta) that produced delta-alpha-delta hybrid genes which differ in the location of breakpoints. This report presents the molecular analysis of HGpMilX, the first example of a reverse alpha-delta-alpha hybrid gene that specifies a newly described phenotype of the Miltenberger complex. A novel restriction fragment unique to the HGpMilX gene was detected by Southern blot hybridization. The structure of the genomic region encoding the entire extracellular domain of the MilX protein was determined. Nucleotide sequencing of amplified genomic DNA showed that a silent segment of the HGpB(delta) gene had been transposed to replace the internal part of exon III in the HGpA(alpha) gene, thereby resulting in the formation of the MilX allele with an alpha-delta-alpha configuration. The proximal alpha-delta breakpoint was found to be flanked by a direct repeat of the acceptor splice site, whereas the distal delta-alpha breakpoint was localized to a palindromic region. This DNA rearrangement, with a minimal transfer of 16 templated nucleotides and a single mutation of untemplated adenyl nucleotide, not only created two intraexon hybrid junctions but transactivated the expression of a new stretch of amino acid residues in the MilX protein. Such a segment replacement may have occurred through the directional transfer from one duplex to the other via the mechanism of gene conversion. The occurrence of HGpMilX as another hybrid derived from parts of parent genes underlines the role of the recombinational "hotspot" in the generation of allelic diversity in the glycophorin family.

Miltenberger class IX of the MNS blood group system.

Mi.IX is a new phenotype in the Miltenberger series of the MNS blood group system with a frequency of 0.43% in Denmark. Mi.IX red cells are Mur+ but do not express any of the other established Miltenberger determinants. They react with a new antibody, anti-DANE, which defines a determinant present on Mi.IX cells but not on cells of other Miltenberger phenotypes. Four Mi.IX propositi have been found. Their families show that MiIX is inherited with a MS complex (lod score 3.69 at theta = 0.00) which produces a trypsin-resistant M antigen. DANE has been allotted the ISBT number 002032 (MNS32). Serological and immunochemical studies with human and monoclonal antibodies to various determinants on glycophorin A (GPA) suggest that Mi.IX is associated with an aberrant GPA molecule that lacks the trypsin cleavage site at amino-acid residue 39, retains the chymotrypsin cleavage site at residue 34 and has an apparent Mr of about 1,000 less than normal GPA. It is proposed that this Mi.IX molecule has an amino acid and possibly also a glycosylation change in the region of amino-acid residues 35-39.

Elution of anti-Zwa (-PIA1) from autologous platelets after normalization of platelet count in post-transfusion purpura.

In two patients suffering from post-transfusion purpura, anti-Zwa was demonstrated in either eluates from autologous platelets harvested during the remission phase and after normalization of the platelet count. In both patients, platelet-associated immunoglobulin (PAIg) was demonstrated during the acute phase. PAIg disappeared concomitantly with recovery and, thus, seems to be associated with the thrombocytopenia. These data support the assumption that immunocomplexes are adsorbed nonspecifically to the platelets and cause destruction of autologous platelets lacking the corresponding antigen. An association between the IgG3 subclass of anti-Zwa antibodies and the destruction of autologous platelets was also seen.

A case of cold haemoglobinuria with later sarcoidosis. Treatment with plasmapheresis and immunosuppressiva.

A case of severe but transient haemolytic disease occurring after a febrile episode is described. The thermal amplitude of the haemolysin was high during the acute phase, since the autoantibody fixed complement at 31 degrees C. After 4 months complement fixation could exclusively be demonstrated at 4 degrees C. The patient was treated in a room heated to 30-32 degrees C. The treatment consisted of prednisone and azathioprine and during the acute phase plasmapheresis was attempted in order to reduce the antibody concentration. However, the haemolysis decreased when the thermal amplitude of the antibody diminished. 1 year after termination of therapy, she developed sarcoidosis.

Observations on the number of available C antigen sites on red cells.

The number of available C (rh', Rh 2) antigen sites within the Rh system was estimated using trace-labelled antibody. The results, as available sites per red cell, were as follows: CDe/CDe: 45,700-56,400; Cde/Cde: 42,200; CDe/cDE: 25,500-39,700; CwDe/cde: 21,500-40, 000; Cde/cde: 31,100; CDE/cDE: 8,500-9,800; Cde/cde: 7,200. The average equilibrium constant of the anti-C was 0.5 X 10(7) M-1.

Observations of the number of available G (rhG, Rh12) antigen sites on red cells.

The number of available G antigen sites within the rhesus system were estimated using trace-labelled antibody. The results given as available antigen sites per cell were as follows: R1R1: 9,900-12,200; r'r': 8,200-9,700; ryr: 8,600; RNRN: 6,600; r'xr: 6,200; RB1r: 6,200; RZRZ: 5,400; Cdes: 4,800-5,100; R2R2: 3,600-5,800; RORO: 4,500-5,300; R2r 4,200; r''Gr: 1,700-3,600; RB2r: 1,400-1,900. The G antigen is detected in optimal amount on C-positive samples. When D is present without C,the number of G sites is apparently dependent on the expression of all the D antigens within the recognized six categories of D antigens.