Prestorage leukofiltration of whole blood and SAGM blood prevents extracellular bioactive substance accumulation.

OBJECTIVES: Potentially harmful leukocyte- and platelet-derived bioactive substances are accumulated extracellularly during storage of different blood products. Therefore, we studied the effect of prestorage leukocyte filtration on concentrations of bioactive substances in whole blood (WB) and saline-adenine-glucose-mannitol (SAGM) erythrocyte suspension during storage. METHODS: Ten units of WB and 10 units of SAGM blood from 20 blood donors were stored at + 4 degrees C for 24 h. Subsequently, half of every unit was leukocyte-reduced by filtration. The 40 half units (20 filtered and 20 unfiltered) were stored at + 4 degrees C for further 34 days. Samples were collected from all 40 half blood units on day 1, 21 and 35. Total content and extracellular concentration of myeloperoxidase (MPO), eosinophil cationic protein (ECP), histamine and plasminogen activator inhibitor-1 (PAI-1) was analysed by ELISA or RIA methods. RESULTS: In unfiltered WB, the total content of all 4 substances decreased during storage, and extracellular concentrations increased significantly and storage time dependently. Similarly, this was also seen with MPO and ECP in unfiltered SAGM blood. Prestorage filtration of WB resulted in a significant reduction of total content and of extracellular concentrations of all 4 substances as well. Additionally, storage time dependent extracellular accumulation was prevented for all substances. Prestorage filtration of SAGM blood significantly reduced total content and extracellular concentrations of MPO and ECP and prevented storage time dependent extracellular accumulation. Filtered SAGM blood contained significantly lower concentrations of all analysed substances compared to filtered WB. CONCLUSION: Prestorage leukocyte filtration reduces total content of leukocyte- and platelet-derived bioactive substances and prevents the storage time dependent extracellular accumulation of these substances in WB and the partly accumulation in SAGM blood.

Prestorage leukocyte filtration may reduce leukocyte-derived bioactive substance accumulation in patients operated for burn trauma.

Adverse effects of perioperative blood transfusion appear to be storage-time-dependent and may be related to extracellular accumulation of bioactive substances in blood products. In this study the clinical effects of leukofiltered and non-filtered blood products in patients undergoing surgery for burn trauma are investigated. 24 consecutive patients were randomly selected to receive transfusion with non-filtered blood components (group A, n = 12) or similar products, which were prestorage leukofiltered (group B, n = 12). The burn injury was scored using the Bull and Fischer index of age and burn surface area. Histamine, interleukin-6 (IL-6), plasminogen activator inhibitor-1 (PAI-1), eosinophil cationic protein (ECP) and myeloperoxidase (MPO) were analysed in plasma or serum collected from all patients 30 min before skin incision, at skin incision and 5, 10 and 30 min and thereafter every 30 min after skin incision until the grafts were secured by wrapping. Samples were also taken 8 h after skin incision and in the morning of postoperative days 1-5. The amount of blood products transfused from admission until day 5 postoperatively was recorded. All patients were followed until discharge or death. The Bull and Fischer index was comparable in the two groups. Prestorage leukofiltration reduced the amount of blood products required for transfusion significantly (p < 0.05) compared with non-filtered products. The levels of the various bioactive substances changed during and after the operation. In particular, ECP and MPO levels increased significantly (p < 0.05) in group A patients compared with unchanged (ECP) or decreased (MPO) levels in group B patients. IL-6 analyses showed, that the trauma had more severe impact on group B patients than on group A patients. Nevertheless, 4 patients died in group A and 2 in group B; all with a Bull and Fischer index between 1.0 and 2.0. Prestorage leukocyte filtration may reduce transfusion related accumulation of various bioactive substances and the requirement for blood in burn trauma patients.

Effect of heating on extracellular bioactive substances in stored human blood: in vitro study.

BACKGROUND: We have previously shown extracellular accumulation of various leukocyte and platelet-derived bioactive substances in human blood during storage. Release of bioactive substances may be temperature-dependent, and we studied the effect of heating during in vitro transfusion on bioactive substance accumulation in stored human blood. METHODS: Eight units of whole blood and eight units of prestorage leukofiltered whole blood were stored at 4 degrees C for 7 days. Subsequently, the blood from all 16 units was transfused via a blood-heating device, which increased the blood temperature to 37 degrees C at outlet. Samples for enzyme-linked immunosorbent assay or radioimmunoassay analyses of histamine, myeloperoxidase (MPO), eosinophil cationic protein (ECP), and plasminogen activator inhibitor-1 (PAI-1) were drawn from the units at donation, after 7 days of storage just before transfusion, and during the in vitro transfusion. RESULTS: Extracellular concentrations of histamine, MPO, ECP, and PAI-1 were significantly (p < 0.05) increased in nonfiltered whole blood stored for 7 days compared with concentrations in fresh donated blood and in prestorage leukofiltered whole blood stored for 7 days. Heating reduced histamine, MPO, and ECP concentrations significantly (p < 0.05) in nonfiltered whole blood, whereas PAI-1 concentrations increased significantly (p < 0.05). Finally, there was no difference in concentrations of histamine, MPO, ECP, and PAI-1 in samples collected before and after heating of leukofiltered whole blood. CONCLUSIONS: Heating reduces accumulation of extracellular leukocyte-derived bioactive substances in whole blood, whereas it increases platelet-derived substances. Prestorage leukofiltration, however, reduces the obligatory extracellular accumulation of leukocyte and platelet-derived bioactive substances, which in addition is unchanged by heating.

ELISA determination of soluble urokinase receptor in blood from healthy donors and cancer patients.

Measurement of urokinase receptor (uPAR) in tumor extracts has prognostic value, but assay of the soluble uPAR (suPAR) in peripheral blood may offer wider applications in cancer patient management. A tumor extract uPAR ELISA was modified to eliminate nonspecific plasma protein interference, enabling specific detection of suPAR in plasma and sera with >90% recovery of added calibrator. suPAR concentrations in citrate plasma correlated with sera in 93 healthy blood donors (r = 0.84, P <0.0001), with a median value for both of 1.2 microg/L. The plasma median for 19 advanced breast cancer patients was 2.9 microg/L suPAR, and a similar increase was found for 10 advanced colon cancer patients, consistent with release of suPAR from tumors into blood. Repetitive monitoring of suPAR in cancer patients' blood may have value in assessment of prognosis and tumor recurrence.

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.

Bioactive substance accumulation and septic complications in a burn trauma patient: effect of perioperative blood transfusion?

Evidence has emerged that suggests adverse effects to perioperative homologous blood transfusion are related to the age of the blood products. Recently, time-dependent accumulation of bioactive substances in red cell suspensions, standard platelet concentrates and fresh frozen plasma during storage have been shown. The potential adverse effects of these bioactive substances were analysed in a burn trauma patient. A patient with 40 per cent second and third degree burn trauma without other injuries underwent a two-step transplantation operation. Samples for analyses of histamine, eosinophil cationic protein (ECP), eosinophil protein X (EPX), neutrophil myeloperoxidase (MPO) and interleukin 6 (IL-6) were drawn frequently from the patient before, during and after the operations, and from all transfused red cell, platelet and fresh frozen plasma units. Urine was sampled every hour during the first operation for analyses of ECP and EPX excretion. All analyses were performed by ELISA and RIA methods, and results compared to patient outcome. The patient received a total of 48 and 8 SAGM blood, 6 and 0 platelet and 12 and 4 fresh frozen plasma units at the two operations, respectively. Transfused products contained a total of 64.54 nmol and 17.50 nmol histamine, 115518 ng and 25764 ng ECP, 174457 ng and 38770 ng EPX, 6950915 ng and 1505125 ng MPO, and 14740 pg and 5600 pg IL-6 at the two operations, respectively. The accumulation of the substances in patient plasma correlated to postoperative septic reactions, without any disclosure of bacteraemia after the first operation, while the accumulation at the second operation correlated to the septic reaction and Pseudomonas aeruginosa infection. Time-dependent accumulation of bioactive substances in blood products during storage may be related to the development of post-transfusion adverse effects.

Time-dependent, spontaneous release of white cell- and platelet-derived bioactive substances from stored human blood.

BACKGROUND: The mechanisms of the detrimental effects of perioperative allogeneic blood transfusion are still unclear. Previous studies have suggested a higher incidence of adverse effects after the use of blood stored for prolonged time. Therefore, a possible time-dependent release of various white cell- and platelet-derived bioactive substances in stored human red cell suspensions was studied. STUDY DESIGN AND METHODS: Whole blood (6 units), plasma-reduced whole blood (6 units), and saline-adenine-glucose-mannitol blood (6 units) from 18 unpaid, normal blood donors were stored under standard blood bank conditions at 4 degrees C for 35 days. After refrigeration, samples were collected from all blood bags on Days 0, 2, 5, 9, 14, 21, 28, and 35 of storage. Extracellular concentrations of eosinophil cationic protein, eosinophil protein X, plasminogen activator inhibitor 1, myeloperoxidase, and interleukin 6 were analyzed by enzyme-linked immunosorbent assay and radioimmunoassay. The total intracellular and donor plasma levels of these substances also were analyzed at the time of blood donation. RESULTS: Eosinophil cationic protein, eosinophil protein X, and myeloperoxidase increased 10- to 25-fold (p < 0.05) in a time-dependent manner in whole blood, plasma-reduced whole blood, and saline-adenine-glucose-mannitol blood during storage for 35 days. Plasminogen activator inhibitor 1 increased threefold to sixfold (p < 0.05) in whole blood and plasma-reduced whole blood, but not in saline-adenine-glucose-mannitol blood. Interleukin 6 was not detected in either plasma or samples obtained from the blood bags. CONCLUSION: Stored whole blood, plasma-reduced whole blood, and saline-adenine-glucose-mannitol blood may release white cell- and platelet-derived bioactive substances in a time-dependent manner, which may be related to the detrimental effects of perioperative blood transfusions. Therefore, prestorage white cell reduction should be considered for further improvement of red cell suspensions.

Buspirone, a serotonin receptor agonist, increases CD4 T-cell counts and modulates the immune system in HIV-seropositive subjects.

OBJECTIVE: We have previously shown that drugs that decrease intracellular cAMP levels increase/restore the proliferative and cytotoxic capacity of T cells from HIV-seropositive subjects in vitro. Buspirone, a serotonin receptor agonist, indirectly decreases intracellular cAMP levels in T cells and has the same increasing/restoring effect on T-cell proliferation in lymphocytes from HIV-seropositive subjects in vitro. DESIGN: Buspirone was given as a single high dose to six HIV-seropositive subjects, or as continuous medication with increasing dosage over 6 weeks to nine HIV-seropositive subjects, with CD4 T-cell counts of 150-300 x 10(6)/l. RESULTS: Significant increases in CD4 T cells, CD4 percentage and CD4/CD8 ratio were found 1 week after a single high dose of buspirone was administered. With continuous administration, a significant increase in CD4 T cells was observed after 1 and 4 weeks. Serum HIV RNA showed a significant decrease 1 h after a single dose of buspirone was administered. With continuous administration of buspirone, plasma HIV RNA first increased within the first 2 weeks of treatment and then decreased towards and below baseline concurrently with a significant decrease in CD8T cells. The proliferative T-cell response to poke weed mitogen and membrane expression of IL-2R increased significantly during continuous treatment with a significant decrease in expression of HLA-DR on CD8+ T cells. Development of "flu-like' symptoms, so severe that two patients withdrew from the study and two patients ceased medication before time, was a clinical indication of modulation of the immune system by buspirone. CONCLUSION: The study shows that buspirone modulates the immune system and leads to changes in the CD4 and CD8 T-cell numbers, functional capacity, cell maturation and viral load.

Time-dependent histamine release from stored human blood products.

Perioperative transfusion of whole blood has been shown to amplify trauma-induced immunosuppression, which could be attenuated by perioperative administration of histamine2 receptor antagonists. Supernatants from different blood products were, therefore, analysed for histamine content during storage. Whole blood (six units), plasma-reduced whole blood (six units), and plasma- and buffy coat-reduced (saline-adenine-glucose-mannitol) (SAGM) blood (six units) from unpaid healthy donors were stored in the blood bank for 35 days at 4 degrees C. Plasma histamine and total cell-bound histamine content at donation, and histamine concentration in samples drawn from the units on days 0, 2, 5, 9, 14, 21, 28 and 35 were analysed with an enzyme-linked immunosorbent assay. Median plasma histamine concentration was 4.8 (range 1.9-14.3) nmol/l (n = 18). Median total cell-bound histamine content was 417.0 (range 176.0-910.0) nmol/l in whole blood and 475.0 (range 360.0-1560.0) nmol/l in plasma-reduced whole blood, while it was undetectable in SAGM blood. Spontaneous histamine release increased in a time-dependent manner from a median of 6.7 (range 2.2-17.4) nmol/l at the time of storage to 175.0 (range 33.0-485.0) nmol/l at day 35 in whole blood, from 18.8 (range 8.2-38.5) to 328.5 (range 224.0-1137.0) nmol/l in plasma-reduced whole blood, and from 0.5 (range 0.5-1.5) to 2.2 (range 1.4-6.9) nmol/l in SAGM blood. These results show spontaneous histamine release during storage of human blood products which contain full amount of leucocytes, and this may play a significant role in detrimental effects of blood transfusion.

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.

False negative results in anti-HIV ELISA due to insufficient antigen coating of microtitre plates.

Since routine ELISA screening of blood donors for anti-HIV antibodies was introduced, much attention has been given to the specificity and the sensitivity of this assay. Most papers deal with false positive reactions while only a few have taken account of false negative results. We have found that insufficient HIV-antigen coating of the microtitre plates can lead to false negative results. It is essential that the producers of the ELISA test kits use control systems which guarantee sufficient antigen coating.

Structural analysis of glycophorin A from Miltenberger class VIII erythrocytes.

The major human erythrocyte membrane sialoglycoprotein (glycophorin A or MN glycoprotein) was purified from the erythrocytes of two individuals heterozygous for the Mi-VIII gene in the Miltenberger subsystem of the MNSs blood-group system. The complete structure of a tryptic glycopetide from glycophorin A comprising the residues 40-61 was deduced from automated and manual sequence analyses. The Mi-VIII-specific glycophorin A was found to exhibit an arginine----threonine exchange at position 49. The threonine residue was found to be glycosylated. Hemagglutination and hemagglutination inhibition assays demonstrated that one of the Mi-VIII-characteristic antigenic determinants (Anek) is located within the residues 40-61 of glycophorin A. Furthermore, erythrocytes from the two Mi-VIII heterozygotes reacted only weakly with anti-EnaKTsera, suggesting that the Mi-VIII-specific glycophorin A does not express the EnaKT antigen that is located within the positions 46-56 of normal glycophorin A. Our data suggest that the Mi-VIII-specific glycophorin A represents the evolutionary link between normal glycophorin A and the Mi-VIII-specific molecule which exhibits arginine----threonine and tyrosine----serine exchanges at the positions 49 and 52, respectively. Our data also provide an explanation for the close serological similarity between Mi-VII and Mi-VIII erythrocytes.

Cost-effectiveness of introducing a third-generation test for HBsAg in Danish blood donors.

Owing to the low incidence of hepatitis B in Denmark, screening of blood donors for HBsAg has mostly been done by immunoelectroosmophoresis (IEOP). The purpose of the present study was to carry out a cost-effectiveness analysis prior to the introduction of a third-generation test for HBsAg in Danish blood donors. The analysis was performed on data from a subsequent screening of 48 750 blood units by radioimmunoassay (RIA) 3 weeks after donation. The RIA-pos., IEOP-neg. blood donors identified in the study were evaluated by a follow-up examination, and the recipients of RIA-pos., IEOP-neg. blood units were monitored for up to 9 months as to the development of acute hepatitis B. The study shows that the estimated cost for each prevented case of transfusion-associated hepatitis B in Denmark is US$ 1100 when screening donors not previously tested by a third-generation technique, and US$ 240 000 when screening donors tested before by this technique.

Screening of Danish blood donors for hepatitis B surface antigen using a third generation technique.

The profit to be gained by testing Danish blood donors for hepatitis B surface antigen (HBsAg) with a third generation technique instead of the currently used immunoelectrophoresis was investigated by additional screening of 48 750 blood units by radioimmunoassay three weeks after donation. Twenty nine units were positive for HBsAg on radioimmunoassay (0.059%). Only six of these were found by immunoelectrophoresis (0.012%). Most of the 23 donors positive on radioimmunoassay and negative on immunoelectrophoresis were healthy carriers of HBsAg (20) or had asymptomatic chronic liver disease (two). One donor had acute hepatitis B. Fifteen of the 23 blood units were transfused. The 15 recipients were monitored biochemically and serologically for up to nine months. One recipient developed fulminant hepatitis B, three developed acute hepatitis B, and one became a healthy carrier of HBsAg. All these patients had received blood from healthy carriers of HBsAg. Two recipients were immunised against HBsAg, and in one patient no seroconversion was observed. The remaining recipients died soon after transfusion or were protected by antibodies to HBsAg that had been present before the transfusion. Testing of Danish blood donors using a third generation technique identified a substantial number of donors positive for HBsAg overlooked by immunoelectrophoresis. Most of these donors were healthy carriers of HBsAg. Blood taken from such carriers is highly infectious when transfused, probably because of the large amount of material transmitted.

Primary immunization-like response without hepatitis following transfusion of HBeAg-positive blood.

An accidental transfusion of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) positive whole blood was given to a 19-yr-old male, bleeding after tonsillectomy. Serum obtained from the patient before the transfusion revealed no hepatitis B antigens or antibodies. After the transfusion the patient became HBsAg-positive, cleared this antigen and developed antibodies to both HBsAg and HBeAg. The transfusion blood was positive for total antibody and IgM antibody to hepatitis B core antigen (HBcAg). The patient's blood became positive for these antibodies after the transfusion, but with declining titres. Liver tests were normal through the entire follow-up. The serological and clinical course suggests immunisation to passively transferred antigens without hepatitis.

Identification of a cold agglutinin with anti-Pr3 specificity.

An apparent monoclonal IgM, kappa-cold agglutinin with anti-Pr specificity was detected in the serum of a 38-year old woman. Haemagglutination inhibition studies disclosed the subspecificity anti-Pr3, since the cold agglutinin was strongly inhibited by carbodiimide-treated erythrocyte glycoproteins. Studies with animal cells showed that the corresponding Pr3 determinant was found only on human red cells. This is in contrast to earlier findings and indicates that distinct fine specificities within anti-Pr3 antibodies might well exist.