Plasma temperature during methylene blue/light treatment influences virus inactivation capacity and product quality.

BACKGROUND: Photodynamic treatment using methylene blue (MB) and visible light is in routine use for pathogen inactivation of human plasma in different countries. Ambient and product temperature conditions for human plasma during production may vary between production sites. The influence of different temperature conditions on virus inactivation capacity and plasma quality of the THERAFLEX MB-Plasma procedure was investigated in this study. METHODS: Plasma units equilibrated to 5 ± 2°C, room temperature (22 ± 2°C) or 30 ± 2°C were treated with MB/light and comparatively assessed for the inactivation capacity for three different viruses, concentrations of MB and its photoproducts, activity of various plasma coagulation factors and clotting time. RESULTS: Reduced solubility of the MB pill was observed at 5 ± 2°C. Photocatalytic degradation of MB increased with increasing temperature, and the greatest formation of photoproducts (mainly azure B) occurred at 30 ± 2°C. Inactivation of suid herpesvirus, bovine viral diarrhoea virus and vesicular stomatitis virus was significantly lower at 5 ± 2°C than at higher temperatures. MB/light treatment affected clotting times and the activity of almost all investigated plasma proteins. Factor VIII (-17·7 ± 8·3%, 22 ± 2°C) and fibrinogen (-14·4 ± 16·4%, 22 ± 2°C) showed the highest decreases in activity. Increasing plasma temperatures resulted in greater changes in clotting time and higher losses of plasma coagulation factor activity. CONCLUSIONS: Temperature conditions for THERAFLEX MB-Plasma treatment must be carefully controlled to assure uniform quality of pathogen-reduced plasma in routine production. Inactivation of cooled plasma is not recommended.

Enlargement of the WHO international repository for platelet transfusion-relevant bacteria reference strains.

BACKGROUND AND OBJECTIVES: Interventions to prevent and detect bacterial contamination of platelet concentrates (PCs) have reduced, but not eliminated the sepsis risk. Standardized bacterial strains are needed to validate detection and pathogen reduction technologies in PCs. Following the establishment of the First International Reference Repository of Platelet Transfusion-Relevant Bacterial Reference Strains (the 'repository'), the World Health Organization (WHO) Expert Committee on Biological Standardisation (ECBS) endorsed further repository expansion. MATERIALS AND METHODS: Sixteen bacterial strains, including the four repository strains, were distributed from the Paul-Ehrlich-Institut (PEI) to 14 laboratories in 10 countries for enumeration, identification and growth measurement on days 2, 4 and 7 after low spiking levels [10-25 colony-forming units (CFU)/PC bag]. Spore-forming (Bacillus cereusPEI-B-P-07-S, Bacillus thuringiensisPEI-B-P-57-S), Gram-negative (Enterobacter cloacaePEI-B-P-43, Morganella morganiiPEI-B-P-74, PEI-B-P-91, Proteus mirabilisPEI-B-P-55, Pseudomonas fluorescensPEI-B-P-77, Salmonella choleraesuisPEI-B-P-78, Serratia marcescensPEI-B-P-56) and Gram-positive (Staphylococcus aureusPEI-B-P-63, Streptococcus dysgalactiaePEI-B-P-71, Streptococcus bovisPEI-B-P-61) strains were evaluated. RESULTS: Bacterial viability was conserved after transport to the participating laboratories with one exception (M. morganiiPEI-B-P-74). All other strains showed moderate-to-excellent growth. Bacillus cereus, B. thuringiensis, E. coli, K. pneumoniae, P. fluorescens, S. marcescens, S. aureus and S. dysgalactiae grew to >106 CFU/ml by day 2. Enterobacter cloacae, P. mirabilis, S. epidermidis, S. bovis and S. pyogenes achieved >106 CFU/ml at day 4. Growth of S. choleraesuis was lower and highly variable. CONCLUSION: The WHO ECBS approved all bacterial strains (except M. morganiiPEI-B-P-74 and S. choleraesuisPEI-B-P-78) for repository enlargement. The strains were stable, suitable for spiking with low CFU numbers, and proliferation was independent of the PC donor.

Effect of increased agitation speed on pathogen inactivation efficacy and in vitro quality in UVC-treated platelet concentrates.

BACKGROUND AND OBJECTIVES: Pathogen inactivation technologies require continuous development for adjustment to different blood components and products. With Theraflex UV-Platelets, a system using shortwave ultraviolet C (UVC) light (254 nm), efficient mixing of platelet concentrates (PCs) during UVC treatment is essential to ensure homogeneous illumination of the blood components. In this study, we investigated the impact of increasing the agitation speed during UVC treatment on pathogen inactivation capacity and platelet quality. MATERIAL AND METHODS: The pathogen inactivation efficacy of UVC treatment was evaluated at two agitation speeds (110 vs. 180 rpm) using four different transfusion-relevant bacteria strains and three model viruses. Using a pool-and-split design, the in vitro quality of buffy coat-derived PCs stored in SSP+ additive solution for up to 7 days was assessed in UVC-treated PCs agitated at either 110 rpm (standard speed) or 180 rpm (increased speed) and in untreated controls. RESULTS: The higher agitation speed improved bacterial inactivation but did not influence viral inactivation. Metabolic activity (glucose consumption and lactate accumulation) in UVC-treated platelets was slightly higher than in untreated controls. Increases in parameters such as CD62P expression and annexin A5 binding indicated moderate activation of UVC-treated platelets. Quality variables for UVC-treated platelets agitated at standard vs. increased agitation speed were comparable. CONCLUSION: The mixing rate during illumination may be a process parameter for further development of UVC-based pathogen inactivation procedures for PLT concentrates.

Challenge study of the pathogen reduction capacity of the THERAFLEX MB-Plasma technology.

BACKGROUND AND OBJECTIVES: Although most pathogen reduction systems for plasma primarily target viruses, bacterial contamination may also occur. This study aimed to investigate the bacterial reduction capacity of a methylene blue (MB) treatment process and its virus inactivation capacity in lipaemic plasma. MATERIALS AND METHODS: Bacterial concentrations in plasma units spiked with different bacterial strains were measured before and after the following steps of the THERAFLEX MB-Plasma procedure: leucocyte filtration, MB/light treatment and MB filtration. Virus inactivation was investigated for three virus types in non-lipaemic, borderline lipaemic and highly lipaemic plasma. RESULTS: Leucocyte filtration alone efficiently eliminated most of the tested bacteria by more than 4 logs (Staphylococcus epidermidis and Staphylococcus aureus) or to the limit of detection (LOD) (≥ 4.8 logs; Escherichia coli, Bacillus cereus and Klebsiella pneumoniae). MB/light and MB filtration further reduced Staphylococcus epidermidis and Staphylococcus aureus to below the LOD. The small bacterium Brevundimonas diminuta was reduced by 1.7 logs by leucocyte filtration alone, and to below the LOD by additional MB/light treatment and MB filtration (≥ 3.7 logs). Suid herpesvirus 1, bovine viral diarrhoea virus and human immunodeficiency virus 1 were efficiently inactivated by THERAFLEX MB-Plasma, independent of the degree of lipaemia. CONCLUSION: THERAFLEX MB-Plasma efficiently reduces bacteria, mainly via the integrated filtration system. Its virus inactivation capacity is sufficient to compensate for reduced light transparency due to lipaemia.

Tolerance of platelet concentrates treated with UVC-light only for pathogen reduction--a phase I clinical trial.

BACKGROUND: The THERAFLEX UV-Platelets pathogen reduction system for platelet concentrates (PCs) operates with ultraviolet C light (UVC; 254 nm) only without addition of photosensitizers. This phase I study evaluated safety and tolerability of autologous UVC-irradiated PCs in healthy volunteers. METHODS: Eleven volunteers underwent two single (series 1 and 2) and one double apheresis (series 3). PCs were treated with UVC, stored for 48 h and retransfused in a dose-escalation scheme: 12·5, 25% and 50% of a PC (series 1); one complete PC (series 2); two PCs (series 3). Platelet counts, fibrinogen, activated partial thromboplastin time, prothrombin time, D-dimer, standard haematology, temperature, heart rate, blood pressure and clinical chemistry parameters were measured. One- and 24-h corrected count increments were determined in series 2 and 3. Platelet-specific antibodies were assessed before and at the end of the study. RESULTS: Neither adverse reactions related to transfusions nor antibodies against UVC-treated platelets were observed. Corrected count increments did not differ between series 2 and 3. CONCLUSIONS: Repeated transfusions of autologous UVC-treated PCs were well tolerated and did not induce antibody responses in all volunteers studied. EudraCT No. 2010-023404-26.

Semaphorin 7A inhibits platelet production from CD34+ progenitor cells.

BACKGROUND: The multifunctional protein semaphorin 7A (Sema7A) may have regulatory effects on blood cell differentiation via its receptors ß1-integrin and plexin C1. As thrombocytopenia can be treated with transfusion of ex vivo CD34(+) cell-derived megakaryocytes, we investigated the effect of Sema7A on differentiation of CD34(+) progenitor cells into megakaryocytes and platelets. METHODS: Megakaryocytes and platelets were differentiated with a specific cytokine cocktail (CC) from CD34(+) progenitor cells in the presence or absence of Sema7A. Expression of cell markers CD41, CD42a and CD61 or detection of the activation of the signal mediator focal adhesion kinase (FAK) was performed by flow cytometry, cytokine secretion by Luminex technology, and megakaryocyte cell density and morphology by microscopic studies. Sema7A levels in vivo were assessed by real-time PCR and ELISA in hematological patients undergoing chemotherapy. RESULTS: CD34(+) progenitor cells expressed the receptors for Sema7A. Expression of CD41, CD42a and CD61 was markedly reduced in the presence of Sema7A, after CC-dependent platelet production from CD34(+) progenitor cells. As revealed by microscopic analysis, megakaryocyte cell density was significantly lower in the presence of Sema7A as compared with controls. Blocking of CD29 abrogated the Sema7A-mediated inhibition. Sema7A activated FAK in CD34(+) progenitor cells and significantly increased secretion of the proinflammatory cytokines IL-6, IL-8 and GM-CSF. Finally, Sema7A levels were up-regulated in 50% of patients after chemotherapy. CONCLUSIONS: Sema7A markedly reduces the production rates of megakaryocytes and platelets from CD34(+) progenitor cells. Hence, up-regulation of Sema7A may be a major risk factor for a reduced platelet repopulation after hematopoietic stem cell transplantation.

Menopausal status affects the susceptibility of stored RBCs to mechanical stress.

The mechanical fragility index (MFI) is an in vitro measure of sublethal injury to RBCs. In our previous experiments, we demonstrated that an increase in sublethal injury (increasing MFI) was a component of the RBC storage lesion, and that the MFI was significantly higher amongst the RBC units from male donors compared to pre-menopausal female donors during storage. It was hypothesized that hormonal or menstrual factors contributed to this difference. In this study, we found that RBC units donated by post-menopausal women demonstrated an MFI that was significantly higher than those donated by pre-menopausal women throughout storage.

Regulation of HLA class II expression prevents allogeneic T-cell responses.

Human leukocyte antigen (HLA) class II molecules are polymorphic heterodimers that present peptides to CD4+ T-cells. The HLA-DM molecule contributes to assemble HLA class II-peptide complexes. We investigated the effect of silencing either HLA-DR or HLA-DM expression in the allogeneic T-cell responses. The delivery of HLA-DR- or HLA-DM-specific short hairpin RNAs (shRNAs) in a monocytic cell line caused a decrease by up to 85% and 75% at the respective mRNA level. Allogeneic T-cells stimulated with HLA-DM-silenced monocytes decreased to 30% granzyme B mRNA and interferon gamma (IFN-γ) production in comparison with T-cells stimulated with monocytes expressing a non-specific shRNA. By contrast, HLA-DR-silenced monocytes did not induce proliferation, up-regulation of granzyme B mRNA levels or high IFN-γ secretion by allogeneic T-cells vs HLA-DR expressing cells. Direct targeting of HLA-DR expression prevented more efficiently an allogeneic T-cell response in comparison with the knockdown of the expression of HLA-DM molecules. Silencing the expression of HLA-DR molecules might contribute to the development of new allogeneic cell-based therapeutic approaches.

The use of the mechanical fragility test in evaluating sublethal RBC injury during storage.

BACKGROUND: The mechanical fragility index (MFI) is an in vitro measurement of the extent of RBC sublethal injury. Sublethal injury might constitute a component of the RBC storage lesion, thus the MFI was determined serially during routine RBC storage. METHODS: Leucoreduced AS-5- and SAGM-preserved RBCs were stored under routine blood bank conditions. The mechanical fragility (MF) of each unit was serially measured during storage. RESULTS: For both AS-5 and SAGM units, male and female RBCs demonstrated statistically significant increases in the MFI during storage. The MFI was significantly lower in AS-5 units compared to SAGM units throughout storage. Female RBCs had significantly lower MFI vs. male RBCs in both AS-5 and SAGM units at all times. No significant differences in MFI were observed between ABO groups for both genders for AS-5 RBCs. CONCLUSIONS: The MF of RBCs increases during storage. Both gender and preservation solution influenced the MFI; however, the male:female MFI ratios were similar at all time-points and remained stable, suggesting that gender-based biological differences exist independent of storage solution. The MF could be a useful test for evaluating the effect of novel interventions intended to mitigate the susceptibility of RBCs to sublethal injury during storage.

SERF: a new antigen in the Cromer blood group system.

The Cromer blood group system consists of eight high incidence and three low incidence antigens carried on decay-accelerating factor (DAF). This report describes the identification and characterization of a new Cromer high incidence antigen, named SERF. Sequence analyses of DNA from a Thai female whose serum contained the alloantibody to a high incidence antigen in the Cromer blood group system (anti-SERF) and from her two children were performed. Reverse transcriptase-polymerase chain reaction (RT-PCR) and sequence analysis on cDNA from the proband and PCR-restriction fragment length polymorphism analysis on DNA from Thais were also performed. To map the epitope, DAF deletion mutants were tested by immunoblotting with anti-SERF. Sequence analysis revealed a substitution of 647C>T in exon 5 DAF in the proband. The proband's two children and two of 100 Thais were heterozygotes 647C/T. Analysis using DAF deletion mutants revealed the antigenic determinant to be within short consensus repeat 3 (SCR3), which is encoded by exon 5. This study describes a novel high incidence antigen (SERF) in the Cromer blood group system characterized by the amino acid proline at position 182 in SCR3 of DAF. The SERF-negative proband has a substitution mutation that predicts for leucine at this position. SERF has been provisionally assigned the International Society of Blood Transfusion number 021.012 (CROM 12).

Acute haemolytic transfusion reactions due to weak antibodies that in vitro did not seem to be clinically significant.

BACKGROUND AND OBJECTIVES: In the present article, we report on two patients with acute haemolytic transfusion reactions (AHTRs), and whom we were unable to transfuse, owing to alloantibodies that in vitro did not seem to be clinically significant. MATERIALS AND METHODS: The patients were a 67-year-old male and a 64-year-old female, both of whom developed antibodies to red blood cells (RBCs) after repeat blood transfusions. Serological analyses were carried out using standard techniques. RESULTS: Both patients developed an AHTR of the intravascular type following blood transfusions. Serological re-examination revealed weakly reactive alloantibodies with anti-JMH specificity in one patient, and with unclear specificity in the second. Rechallenging the patients with 15-30 ml of packed RBCs caused AHTRs, and blood transfusion became impossible in both cases. CONCLUSION: Weak alloantibodies that in vitro do not seem to be clinically significant may cause severe AHTRs.

Rapid detection of autoantibodies to dsDNA with the particle gel immunoassay (ID-PaGIA).

OBJECTIVE: To describe a new particle agglutination test for the detection of autoantibodies to double stranded DNA (dsDNA). PATIENTS AND METHODS: Serum samples were collected from 40 unselected healthy blood donors and 200 patients with systemic lupus erythematosus (SLE) or a positive antinuclear antibody screen, or both. The samples were tested in the presence of red high density polystyrene particles coated with purified human dsDNA using the gel technique (Micro Typing System, ID-PaGIA, particle gel immunoassay). The results were compared with those obtained by the two standard anti-dsDNA antibody detection methods, Crithidia luciliae immunofluorescence test (CLIF) and enzyme linked immunosorbent assay (ELISA). RESULTS: The three anti-dsDNA assays exhibited an overall agreement of 87% and significant correlation with each other (p<0.0001). In the SLE group (n=71), 45 patients (63%) were found to be positive by ID-PaGIA compared with only 11/129 (9%) patients in the non-SLE group. Thus the ID-PaGIA had a sensitivity of 63%, and a specificity of 92% for SLE. In comparison, the standard detection methods showed sensitivities of 62% (CLIF) and 70% (ELISA) and specificities of 99% (CLIF) and 84% (ELISA) for SLE. Anti-dsDNA reactivity in the agglutination assay correlated closely with the quantities of antibody obtained by CLIF (r=0.81, p<0.0001) and ELISA (r=0.73, p<0.0001). CONCLUSIONS: The new particle gel agglutination test is a sensitive and specific immunoassay. It is a simple test procedure that might be well suited as a rapid screening method.

Fatal acute diclofenac-induced immune hemolytic anemia.

We report the case of a 72-year-old woman who developed fatal immune hemolytic anemia with multisystem organ failure and shock caused by diclofenac-dependent red blood cell autoantibodies. The patient described dramatically illustrates the potential severity of this adverse reaction and emphasizes the need for increased awareness of this complication of drug therapy.

Donor-derived alloantibodies and passenger lymphocyte syndrome in two of four patients who received different organs from the same donor.

BACKGROUND: Reported here is the occurrence of RBC alloimmunization in two of four patients who received different organs from an immunized donor. STUDY DESIGN AND METHODS: The donor, a 58-year-old woman, was group O D+, K-, and Fy(a-). Initially, her serum contained only a K antibody. After blood transfusion, a second antibody (anti-Fy(a)) could also be identified. The liver was given to a group O D+, K-, Fy(a+) patient; the pancreas and one kidney to a group O D+, K-, Fy(a+) patient; the heart to a group A D+, K-, Fy(a-) patient; and the other kidney to a group B D+, K-, Fy(a+) patient. RBC grouping and antibody screening were performed by standard techniques. Lymphoid microchimerism in the peripheral blood of the recipients was analyzed by flow cytometry and nested PCR. RESULTS: None of the recipients had irregular RBC alloantibodies at the time of transplantation. After the transplant, anti-K became detectable in the serum of the liver recipient, and anti-Fy(a) could be eluted from the RBCs of the liver recipient and the pancreas-kidney recipient. The latter patient also developed mild hemolysis, and his Hb dropped to 8 g per dL on posttransplant Day 9. Donor-derived lymphocytes were detectable by flow cytometry in the peripheral blood of the liver recipient and the pancreas-kidney recipient until Days 8 and 63, respectively, whereas no lymphoid chimerism could be demonstrated in the heart recipient. PCR chimerism analyses were positive in all three recipients over the whole observation period of 97 postoperative days. CONCLUSION: The amount of cotransplanted lymphoid tissue may correlate with the extent of peripheral lymphoid microchimerism and the antibody-formation capacity in solid organ transplantation.

Ceftriaxone-induced immune haemolysis: two case reports and a concise review of the literature.

BACKGROUND AND OBJECTIVES: In this report, we will describe the occurrence of intravascular immune haemolytic anaemia (IHA) associated with ceftriaxone and/or its metabolites in two of our patients. Serological examinations were carried out to demonstrate and characterise the causative antibodies. The findings of all previously reported cases will also be discussed. MATERIALS AND METHODS: Direct antiglobulin tests (DAT) and indirect antiglobulin tests were performed according to standard procedures. Tests for drug-dependent antibodies were performed in the presence and absence of the target drugs and their ex vivo antigens (in the urine of patients treated with the drugs). RESULTS: Ceftriaxone-related haemolysis resulted in the death of one of our patients (patient 2), and caused acute renal failure in the other (patient 1). The DATwas strongly positive for anti-C3d and anti-IgG in one case (patient 2), and for anti-C3d alone in the other (patient 1). The serum of patient 1 reacted with red blood cells only in the presence of ex vivo antigens, while that of patient 2 reacted positively to native ceftriaxone and its ex vivo antigen. In the latter patient, the antibodies appeared to cross-react with native cefotaxime whereas, in the first patient, they weakly cross-reacted only with the ex vivo antigens of cefotaxime and cefpodoxime proxetil. CONCLUSION: Ceftriaxone and/or its trace metabolites may induce life-threatening IHA in children and adults. Serological work-up must include tests to determine the cross-reactivity of ceftriaxone-dependent antibodies to avoid immune haemolysis due to administration of structurally related cephalosporins in affected patients.

Vaccination-associated immune hemolytic anemia in two children.

BACKGROUND: Two children in whom acute autoimmune hemolytic anemia (AIHA) developed after vaccination were studied. CASE REPORTS: The children were a 20-month-old girl and a 21-month-old boy. The diagnosis of AIHA was made in accordance with established criteria (hemolysis, positive DAT, and lack of other reasons for the hemolysis). Serologic tests were performed according to standard technique. RESULTS: The girl experienced two attacks of hemolysis. The first episode occurred 2 weeks after oral polio vaccination, and the second episode was observed 7 months later, when she received a simultaneous vaccination against mumps, rubella, and measles. The DAT was strongly positive with anti-C3d. No autoantibodies were detectable in either episode. The boy experienced acute hemolysis a few days after a simultaneous revaccination against diphtheria-pertussis-tetanus, Haemophilus influenzae, hepatitis B, and polio. The DAT using anti-IgG was strongly positive, and the DAT performed with anti-C3d was weakly positive. CONCLUSION: Vaccination-induced AIHA resembles those forms of AIHA related to infectious diseases, and it may occur more frequently than has been reported.

[Intraocular silicone oil tamponade. A clinico-pathologic study of 36 enucleated eyes]. / Intraokuläre Silikonöltamponade. Eine klinisch-pathologische Studie an 36 unukleierten Augen.

BACKGROUND: Previous histological studies have shown that intraocular silicone oil induces irreversible changes in ocular tissues, especially the retina. The purpose of this study was to analyze, in a larger group of enucleated eyes, changes in intraocular tissue after silicone oil injection, dependent on intraocular pressure, how long the oil was in the eye, and the viscosity of intraocular silicone oil. PATIENTS AND METHODS: We did histological examinations on 36 enucleated globes with intraocular silicone oil after vitreoretinal surgery and compared them with 68 enucleated globes treated with buckle and encircling band using immunohistochemistry (n = 5) and electron microscopy (n = 7). For statistical evaluation we used the chi(2) test and analysis of variance. RESULTS: After silicone oil injection we observed a more pronounced reduction in corneal endothelial cells (58%), more frequent closed chamber angle (86%), atrophy of the ciliary body (80%) (P < 0.05), proliferative vitreoretinopathy (89%), and glaucomatous atrophy of the optic nerve (56%) (P < 0.01). The retinae showed independent of the use of silicone oil a loss of inner and outer segments of photoreceptors and of ganglion cells and thinning and rareficaton of all other retinal layers. Globes with silicone oil revealed vacuoles both free and incorporated by macrophages in all layers of the retina. Similar vacuoles were seen in the optic nerve, choroid, retinal pigment epithelium, ciliary body, iris, chamber angle and the corneal endothelium. Silicone oil vacuoles were seen in the retina and optic nerve by 1 month after surgery in two eyes with high intraocular pressure (42 mmHg). Six of eight eyes with normal intraocular pressure levels showed retinal vacuoles, 3 of them after 2 months. Vacuoles in the optic nerve were found in eight of nine eyes with intraocular instillation of 1000 mPa silicone oil. There was no clinicohistopathological correlation between the presence of vacuoles in the retina or optic nerve and the duration and viscosity of intraocular silicone oil. CONCLUSIONS: This study suggests that vacuoles in eyes with silicone oil instillation can be found in the retina after 4 weeks. The period of intraocular silicone oil should be limited to 3-6 months.