The compound effect of irradiation and familial pseudohyperkalemia on potassium leak from red blood cells.

BACKGROUND: Familial pseudohyperkalemia (FP) is a rare asymptomatic condition characterized by an increased rate of potassium leak from red blood cells (RBC) on refrigeration. Gamma irradiation compromises RBC membrane integrity and accelerates potassium leakage. Here, we compared the effect of irradiation, applied early or late in storage, on FP versus non-FP RBC. STUDY DESIGN: Five FP and 10 non-FP individuals from the National Institute for Health Research Cambridge BioResource, UK, and three FP and six non-FP individuals identified by Australian Red Cross Lifeblood consented to the study. Blood was collected according to standard practice in each center, held overnight at 18-24°C, leucocyte-depleted, and processed into red cell concentrates (RCC) in Saline Adenine Glucose Mannitol. On Day 1, RCC were split equally into six Red Cell Splits (RCS). Two RCS remained non-irradiated, two were irradiated on Day 1 and two were irradiated on Day 14. RBCs were tested over cold storage for quality parameters. RESULTS: As expected, non-irradiated FP RCS had significantly higher supernatant potassium levels than controls throughout 28 days of storage (p < .001). When irradiated early, FP RCS released potassium at similar rates to control. When irradiated late, FP RCS supernatants had higher initial post-irradiation potassium concentration than controls but were similar to controls by the end of storage (14 days post-irradiation). No other parameters studied showed a significant difference between FP and control. DISCUSSION: FP does not increase the rate of potassium leak from irradiated RBCs. Irradiation may cause a membrane defect similar to that in FP RBCs.

X- and gamma-irradiation have similar effects on the in vitro quality of stored red cell components.

BACKGROUND: Blood components are irradiated to inactivate lymphocytes to prevent transfusion-associated graft versus host disease. As there are little data regarding the effects of X-irradiation on red blood cell components (RBCs), the in vitro quality of stored red cells (standard, pediatric, washed, and intra-uterine transfusion [IUT]) following X- or gamma-irradiation was compared. STUDY DESIGN AND METHODS: RBCs were pooled, split, and processed to produce standard (<14 days and < 5 days post-collection), pediatric (<5 days post-collection), washed (<14 days post-collection), or IUT RBCs (<5 days post-collection). Standard RBCs were either X- or gamma-irradiated (n = 10 pairs). A further 10 replicates were prepared by pooling and splitting three matched RBCs (X-, gamma-, and non-irradiated). All other RBCs were either X- or gamma-irradiated (n = 20 pairs). Red cell indices, hemolysis, potassium release, metabolism, microparticles, ATP, and 2,3-DPG were measured pre-irradiation and 6 h, 1, 2, 3, 7, 10, and 14 days post-irradiation, depending on the component type. Data were analyzed using two-way repeated measures ANOVA. RESULTS: There were no significant differences in any in vitro quality measurements, with the exception of marginally higher potassium release in washed, IUT, and RBCs <5 days old (p < .0001) following X-irradiation. Both irradiation types increased generation of microvesicles, particularly in components that were older at the time of irradiation or stored for longer post-irradiation. CONCLUSION: X- and gamma-irradiation have similar effects on the in vitro quality of RBCs, indicating that either technology is suitable for blood component irradiation.

Serum growth factor stability in different eye drop packaging systems during storage.

Serum eye drops (SED) have shown beneficial effects in patients suffering from dry eye syndrome and are manufactured for an increasing number of patients in Australia every year. Previous studies have examined the stability of serum growth factors during storage in either experimental vessels not used as the final packaging system or in eye drop bottles. To ensure the quality and safety of SED product manufactured in Australia, the stability of growth factors in serum packaged into two different systems during storage at different temperatures was examined. Healthy blood donors provided a whole blood donation, from which serum was prepared, diluted to 20% and dispensed into either a tube or a vial packaging system. The stability of growth factors, fibronectin and total protein in tube segments was comparable to matched vials samples during storage at -30 °C, 4 °C, 22 °C and 37 °C, with the exception of EGF and fibronectin in 20% SED stored in tube segments, which were more sensitive to storage conditions at 4 °C and 22 °C when compared to vials. Additionally, the growth factor, fibronectin and total protein concentration in both tube segments and vials was stable during storage at -30 °C for at least 9 months. This study highlights the impact of different manufacturing procedures on serum growth factor stability during storage.

D-immunized blood donors who are female and who possess at least one HLA-DRB1*15 allele show a propensity for high serum RhIG production.

BACKGROUND: D- individuals with previous D-incompatible pregnancies and/or blood transfusions, as well as those who are actively immunized with small-volume D+ red blood cells (RBCs), are stimulated to produce RhIG. Many factors could influence the stimulation of immunoglobulin production in response to foreign antigen (such as antigen immunogenicity and genetic factors), and it is unknown whether genetic markers could potentially identify responder anti-D donors. STUDY DESIGN AND METHODS: Anti-D donors were assigned a responder profile based on their serum RhIG levels (n = 431). A subset of donors (n = 272) had DNA extracted for polymerase chain reaction genotyping assays for target genes in antigen presentation and pathogen recognition receptors (TLR2, TLR4, CD14, FcγRIIA, and the MHC Class II locus HLA-DRB1). Statistical tests for associations between anti-D donor responder profiles and genetic factors were performed. RESULTS: A large proportion of our donors (38.7%) were classified as nonresponder donors, despite receiving multiple D+ RBC immunizations, whereas female sex was significantly associated with an all-responder profile (p < 0.001). The presence of the DRB1*15 allele and absence of the DRB1*04 allele were more likely to be associated with a responder anti-D donor, although not significantly after Bonferroni correction. A combination of the DRB1*15 allele and female sex was significantly associated with an anti-D donor responder profile. CONCLUSION: This study has identified female sex and the HLA-DRB1*15 allele as potentially useful markers that could be used to screen donors before entry into D immunization programs.

Inhibition of 2',5'-oligoadenylate synthetase expression and function by the human cytomegalovirus ORF94 gene product.

The human cytomegalovirus (HCMV) ORF94 gene product has been reported to be expressed during both productive and latent phases of infection, although its function is unknown. We report that expression of pORF94 leads to decreased 2',5'-oligoadenylate synthetase (OAS) expression in transfected cells with and without interferon stimulation. Furthermore, the functional activity of OAS was inhibited by pORF94. Finally, we present evidence of OAS modulation by pORF94 during productive HCMV infection of human fibroblasts. This study provides the first identification of a function for pORF94 and identifies an additional means by which HCMV may limit a critical host cell antiviral response.

Immunomodulation of inflammatory leukocyte markers during intravenous immunoglobulin treatment associated with clinical efficacy in chronic inflammatory demyelinating polyradiculoneuropathy.

OBJECTIVE: The objective of the study was to profile leukocyte markers modulated during intravenous immunoglobulin (IVIg) treatment, and to identify markers and immune pathways associated with clinical efficacy of IVIg for chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) with potential for monitoring treatment efficacy. METHODS: Response to IVIg treatment in newly diagnosed IVIg-naïve and established IVIg-experienced patients was assessed by changes in expression of inflammatory leukocyte markers by flow cytometry. The adjusted INCAT disability and Medical Research Council sum scores defined clinical response. RESULTS: Intravenous immunoglobulin modulated immunopathogenic pathways associated with inflammatory disease in CIDP. Leukocyte markers of clinical efficacy included reduced CD185+ follicular helper T cells, increased regulatory markers (CD23 and CD72) on B cells, and reduction in the circulating inflammatory CD16+ myeloid dendritic cell (mDC) population and concomitant increase in CD62L and CD195 defining a less inflammatory lymphoid homing mDC phenotype. A decline in inflammatory CD16+ dendritic cells was associated with clinical improvement or stability, and correlated with magnitude of improvement in neurological assessment scores, but did not predict relapse. IVIg also induced a nonspecific improvement in regulatory and reduced inflammatory markers not associated with clinical response. CONCLUSIONS: Clinically effective IVIg modulated inflammatory and regulatory pathways associated with ongoing control or resolution of CIDP disease. Some of these markers have potential for monitoring outcome.

Identification of genetic polymorphisms that predict responder/non-responder profiles to the RhD antigen.

BACKGROUND: Regular plasma donors who produce high titre anti-D immunoglobulin (Ig) are overseen by the Australian Red Cross Blood Service RhD Program. New donors to the program are immunised with small amounts of RhD-positive RBCs, whilst donors who have developed anti-D due to previous RhD-incompatible blood transfusion or pregnancy are boosted with RhD-positive RBCs to maintain a high level of serum anti-D Ig. A significant proportion of primarily immunised individuals do not respond to RhD immunisation and are therefore unnecessarily exposed to the risks involved in RBC sensitisation. STUDY DESIGN AND METHODS: We genotyped 184 anti-D donors for ∼9000 immunological and inflammatory genetic polymorphisms on an Affymetrix GeneChip, and validated the results with a High-Resolution Melt analysis assay. We built and validated a predictive logistic regression model using High Responder and Non-Responder anti-D donors that incorporated highly-associated polymorphisms and gender. RESULTS: High Responder and Non-Responder profiles in anti-D donors were significantly associated with a shortlist of 13 genetic polymorphisms and sex of the donor. The derivation of a logistic regression model showed an accuracy rate of 92.6% that was subsequently validated as 60.0% with an independent set of donor samples. CONCLUSION: This study has developed a logistic regression model and a genotyping assay that can predict the responder profiles of anti-D donors and could potentially be applied to new donors and transfusion-dependent patients in a clinical setting. Additionally, target polymorphisms identified in immunological genes could help to elucidate the immunomodulatory pathways regulating the immune response to the RhD antigen, and to other RBC antigens.