Rhesus D Antigenic Determinants on Residual Red Blood Cells in Apheresis and Buffy Coat Platelet Concentrates.

BACKGROUND: The level of residual red blood cells (RBCs) in platelet concentrates (PCs) is of interest because of clinical concerns related to alloimmunization to RBC antigens in transfused patients. This work aims at characterizing and quantifying the levels of intact and fragmented RBCs in apheresis (AP-PCs) and buffy coat PCs (BC-PCs) to assess their potential risk for RhD antigen alloimmunization. METHODS: After staining with anti-CD41 (platelets) and anti-CD235a (RBCs) antibodies, the size and density of RhD antigen on intact and fragmented RBCs were analyzed by flow cytometry. RESULTS: Residual RBC counts were 29 ± 22 × 106/unit in AP-PCs and 121 ± 54 × 106/unit in BC-PCs, which correspond to about 3 and 11 µL of RBCs by product, respectively. RhD expression was about 4 times higher on RBC particles in AP-PCs, and these particles contribute to 66 and 75% of the total antigenic load in BC-PCs and AP-PCs, respectively. CONCLUSIONS: Processing methods influence the quantity and nature of contaminating residual RBCs and RBC-derived particles in PCs. The estimation of residual RBCs in these blood products is generally based on measurements of intact RBCs, which might underestimate the risk for alloim-munization in transfused patients. The question of whether these RBC-derived particles can produce an immune response and, thus, should then be taken into consideration for Rh immune prophylactic treatments, remains to be clarified.

Bordetella holmesii Contamination of Platelet Concentrates: Revisiting the Definition of a Positive Culture.

Bacterial contamination remains the most important infectious risk of platelet transfusion. After an initially positive result, a second test is performed on the blood products and the initial culture bottle to confirm the contamination. Based on the blood center's decision algorithm used, results can be either confirmed negative, positive, or indeterminate, or be unconfirmed or discordant. Here, we report the first cases of platelet concentrates contaminated with Bordetella holmesii The in vitro growth characteristics of this unusual contaminant in platelet concentrate were investigated. Two B. holmesii strains isolated from platelet concentrates, as well as a control strain (Serratia marcescens), were spiked into platelet concentrates (PCs) at 1 and 10 CFU/ml. PCs were stored at 20 to 24°C under agitation. Samples were collected on days 2, 3, 4, and 7 for colony count and for bacterial screening using the BacT/Alert 3D system. Two PCs were detected as being positive for B. holmesii However, recultures were negative. In vitro, B. holmesii did not grow but remained detectable in PCs. Its viability diminished rapidly in contact with human plasma. Upon screening using the BacT/Alert 3D system, the majority of products spiked with B. holmesii were negative. This is the first description of PCs contaminated with B. holmesii This bacterium survives in blood products and remains dormant at low concentrations in blood products stored at room temperature, thus making difficult its detection with the BacT/Alert 3D system. The present definition of a true-positive culture of PCs may be overly restrictive for certain bacterial strains.

Pressure Infusion Cuff and Blood Warmer during Massive Transfusion: An Experimental Study About Hemolysis and Hypothermia.

BACKGROUND: Blood warmers were developed to reduce the risk of hypothermia associated with the infusion of cold blood products. During massive transfusion, these devices are used with compression sleeve, which induce a major stress to red blood cells. In this setting, the combination of blood warmer and compression sleeve could generate hemolysis and harm the patient. We conducted this study to compare the impact of different pressure rates on the hemolysis of packed red blood cells and on the outlet temperature when a blood warmer set at 41.5°C is used. METHODS: Pressure rates tested were 150 and 300 mmHg. Ten packed red blood cells units were provided by Héma-Québec and each unit was sequentially tested. RESULTS: We found no increase in hemolysis either at 150 or 300 mmHg. By cons, we found that the blood warmer was not effective at warming the red blood cells at the specified temperature. At 150 mmHg, the outlet temperature reached 37.1°C and at 300 mmHg, the temperature was 33.7°C. CONCLUSION: To use a blood warmer set at 41.5°C in conjunction with a compression sleeve at 150 or 300 mmHg does not generate hemolysis. At 300 mmHg a blood warmer set at 41.5°C does not totally avoid a risk of hypothermia.

Heart valve allograft decontamination with antibiotics: impact of the temperature of incubation on efficacy.

Heart valve allografts are typically processed at 4°C in North America, including the step of antibiotic decontamination. In our own experience with heart valve banking, we often observe persistent positive cultures following decontamination at wet ice temperature. We hypothesized that warmer temperatures of incubation might increase the efficacy of the decontamination procedure. In a first series of experiments, 12 different bacterial species were grown overnight, frozen in standardized aliquots and used directly to inoculate antibiotic cocktail aliquots at 105 colony-forming units (CFU)/ml. The antibiotic cocktail contains vancomycin (50 µg/ml), gentamicin (80 µg/ml) and cefoxitin (240 µg/ml) in Dulbecco's Modified Eagle's Medium. Inoculated aliquots were incubated at 4, 22 and 37°C and CFUs were determined at regular intervals up to 24 h post-inoculation. In a second set of experiments, 10 heart valves were spiked with 5000 CFU/ml and incubated with antibiotics at 4 and 37°C for 24 h. The final rinse solutions of these heart valves were filtered and tested for bacterial growth. After 24 h of incubation, CFUs of all 12 bacterial species were reduced by a factor of only one to two logs at 4°C whereas log reductions of 3.7 and 5.0 or higher were obtained at 22 and 37°C, respectively. Most microorganisms, including Staphylococcus epidermidis, Lactococcus lactis lactis and Propionibacterium acnes survived well the 24-h antibiotic treatment at 4°C (< 1 Log reduction). All 10 heart valves that were spiked with microorganisms had positive final rinse solutions after antibiotic soaking at 4°C, whereas 8 out of 10 cultures were negative when antibiotic decontamination was done at 37°C. These experiments show that a wet ice temperature greatly reduces the efficacy of the allograft decontamination process as microorganisms survived well to a 24-h 4°C antibiotic treatment. This could explain the high rate of positive post-processing cultures obtained with our routine tissue decontamination procedure. Increasing the decontamination temperature from 4 to 37°C may significantly reduce the incidence of post-disinfection bacterial contamination of heart valves.

CD40-activated B cells from patients with systemic lupus erythematosus can be modulated by therapeutic immunoglobulins in vitro.

INTRODUCTION: Aberrant signaling within and between B and T cells, considered to be central in systemic lupus erythematosus (SLE), could depend on enhanced CD40-CD154 activation. As a result, autoreactive B cells, normally anergic, differentiate and secrete antibodies attacking several normal tissues. Thus restorating B cell homeostasis might help control this disease. In this study, two facets of SLE B cells were investigated, namely their in vitro response to CD40-CD154 and the effect of treatment with human immunoglobulins for intravenous use (IVIg). MATERIALS AND METHODS: Blood samples from SLE patients and healthy volunteers were obtained and used to isolate B cells, which were activated through CD40 in the presence or absence of IVIg. The phenotype, proliferation, and differentiation of the SLE B cells were determined and compared with those of control B cells using flow cytometry and standard ELISA. RESULTS: In this model, CD40-activated SLE B cells, as control B cells, proliferated and differentiated and were characterized by the emergence of CD19(lo)CD38(++)CD138(+)CD27(++) cells. IVIg treatment of the CD40-activated SLE B cells resulted in higher differentiation, characterized by increased secretion rates of IgG and IgM, as reported previously for control B cells. CONCLUSIONS: Taken as a whole, such accelerated differentiation of CD40-activated B cells suggests that IVIg may participate in re-equilibration of the antibody repertoire by replacing pathological antibodies by de novo harmless antibodies.

Immunomodulation of human B cells following treatment with intravenous immunoglobulins involves increased phosphorylation of extracellular signal-regulated kinases 1 and 2.

In the treatment of autoimmune diseases, intravenous Igs (IVIg) are assumed to modulate immune cells through the binding of surface receptors. IVIg act upon definite human B cell populations to modulate Ig repertoire, and such modulation might proceed through intracellular signaling. However, the heterogeneity of human B cell populations complicates investigations of the intracellular pathways involved in IVIg-induced B cell modulation. The aim of this study was to establish a model allowing the screening of IVIg signal transduction in human B cell lines and to attempt transposing observations made in cell lines to normal human B lymphocytes. Nine human B cell lines were treated with IVIg with the goal of selecting the most suitable model for human B lymphocytes. The IgG(+) DB cell line, whose response was similar to that of human B lymphocytes, showed reduced IVIg modulation following addition of PD98059, an inhibitor of extracellular signal-regulated protein kinase 1/2 (ERK1/2). The IVIg-induced ERK1/2 phosphorylation was indeed proportional to the dosage of monomeric IVIg used when tested on DB cells as well as Pfeiffer cells, another IgG(+) cell line. In addition, two other intermediates, Grb2-associated binder 1 (Gab1) and Akt, showed increased phosphorylation in IVIg-treated DB cells. IVIg induction of ERK1/2 phosphorylation was finally observed in peripheral human B lymphocytes, specifically within the IgG(+) B cell population. In conclusion, IVIg immunomodulation of human B cells can thus be linked to intracellular transduction pathways involving the phosphorylation of ERK1/2, which in combination with Gab1 and Akt, may be related to B cell antigen receptor signaling.

Comparison of promoter activities for efficient expression into human B cells and haematopoietic progenitors with adenovirus Ad5/F35.

Adenoviral gene transfer into human B lymphocytes and haematopoietic progenitors would allow the characterization of their function on cellular growth, differentiation and survival. Efficient gene expression is however strongly dependent on the promoter used. In this study, we investigated the relative strength of various promoters by following and measuring the expression of the reporter gene EYFP in human peripheral B lymphocytes, cord blood CD34(+) cells and the megakaryocytic cell line M-07e. The murine PGK promoter provided the best level of transgene expression in CD34(+) cells among the four promoters tested, followed closely by the CMV promoter, and to a lesser extend by a CMV promoter with a beta-globin/IgG chimeric intron, whereas the human CD40 promoter provided the lowest levels of expression. In contrast, the strongest promoters in B lymphocytes were the two CMV promoters. Surprisingly, even the best promoters were unable to induce transgene expression in more than 75-80% of the primary B and CD34(+) cells, even though 100% of the cells were infected. Finally and in contrast to retroviruses, only a minority of B lymphocytes and CD34(+) cells were able to induce the transcription of IRES-containing bicistronic expression cassettes from adenovirus.

Efficient gene transfer into normal human B lymphocytes with the chimeric adenoviral vector Ad5/F35.

The failure to efficiently introduce genes into normal cells such as human B lymphocytes limits the characterization of their function on cellular growth, differentiation and survival. Recent studies have shown that a new adenoviral vector Ad5/F35 can efficiently transduce human haematopoietic CD34+ progenitor cells. In this study, we compared the gene transfer efficiencies of the Ad5/F35 vector to that of the parental vector Ad5 in human B lymphocytes. Peripheral blood B cells obtained from healthy individuals were cultured in vitro using CD40-CD154 system. Normal B lymphocytes were infected with replication-defectives Ad5 and Ad5/F35, both containing the GFP reporter gene, and transduction efficiencies were monitored by flow cytometry. Ad5 was highly ineffective, infecting only about 5% of human B lymphocytes. In contrast, Ad5/F35 transduced up to 60% of human B lymphocytes and GFP expression could be detected for up to 5 days post infection. Importantly, physiology of B lymphocytes such as proliferation, viability and antibodies secretion were unaffected following Ad5/F35 transduction. Finally, we observed that memory B lymphocytes were more susceptible to Ad5/F35 infection than naïve B lymphocytes. Thus, our results demonstrate that the adenoviral vector Ad5/F35 is an efficient tool for the functional characterization of genes in B lymphopoiesis.