Vitamin E nanoemulsion activity on stored red blood cells.

BACKGROUND: Stored red blood cells (RBCs) undergo numerous changes that have been termed RBC storage lesion, which can be related to oxidative damage. Vitamin E is an important antioxidant, acting on cell lipids. Thus, this study aimed to investigate vitamin E activity on stored RBCs. METHODS: We prepared a vitamin E nanoemulsion that was added to RBC units and stored at 4 °C. Controls, without vitamin E, were kept under the same conditions. Reactive oxygen species (ROS) production was monitored for up to 35 days of storage. RBC elasticity was also evaluated using an optical tweezer system. RESULTS: Vitamin E-treated samples presented a significant decrease in ROS production. Additionally, the elastic constant for vitamin E-treated RBCs did not differ from the control. CONCLUSION: Vitamin E decreased the amount of ROS in stored RBCs. Because vitamin E acts on lipid oxidation, results suggest that protein oxidation should also be considered a key factor for erythrocyte elastic properties. Thus, further studies combining vitamin E with protein antioxidants deserve attention, aiming to better preserve overall stored RBC properties.

The use of a potential novel tool in virtual crossmatching for platelet transfusion in platelet refractoriness.

BACKGROUND AND OBJECTIVES: Transfusion support for immune-mediated platelet refractoriness (PR) is clinically challenging, technically laborious and costly. The development of 'EpHLA/EpVix software' has been used successfully to select kidney donors. Here, we sought to evaluate this new software as a tool for platelet virtual crossmatch (VxM). MATERIALS AND METHODS: This is a prospective study from 2007 to 2014 of PR patients in a tertiary hospital. Platelet components selected by HLAMatchmaker program were crossmatched by EpHLA/EpVix, anti-human globulin complement-dependent lymphocytotoxicity test (AHG-CDC), flow cytometry platelet crossmatch (FCxM) and then compared. Effectiveness of platelet components transfused was evaluated by CCI. RESULTS: Ninety-seven crossmatched platelet transfusions for 27 patients were enrolled. Partial matches were analysed for 75 transfusions by the 3 methods, and 22% showed discrepant results among the assays. After further analysis, data showed that all divergent cases could be explained by HPA alloimmunization, prozone effect (FCxM), low sensitivity of AHG-CDC and possible interference in FCxM/AHG-CDC assays. Notably, sensitivity and specificity of VxM analysis was excellent (100%). Satisfactory CCI counts were obtained for the majority (22/30) of the transfusions. CONCLUSION: The new EpHLA/EpVix method showed to be effective, feasible and fast for VxM at no cost and able to minimize labour on donor identification. However, platelet crossmatching may be a necessary step because EpHLA/EpVix does not formally exclude HPA alloimmunization.

Bartonella henselae survives after the storage period of red blood cell units: is it transmissible by transfusion?

Bartonella henselae is the agent of cat scratch disease and bacillary angiomatosis. Blood donors can be asymptomatic carriers of B. henselae and the risk for transmission by transfusion should be considered. The objective of this study was to demonstrate that B. henselae remains viable in red blood cell (RBC) units at the end of the storage period. Two RBC units were split into two portions. One portion was inoculated with B. henselae and the other was used as a control. All units were stored at 4 degrees C for 35 days. Aliquots were collected on a weekly basis for culture in a dish with chocolate agar, ideal for the cultivation of this agent. Samples were collected on days 1 and 35 and taken for culture in Bact/Alert R blood culture bottles. Aliquots taken simultaneously were fixed in Karnovsky's medium for subsequent electron microscopy evaluation. Samples from infected bags successfully isolated B. henselae by chocolate agar culture, although Bact/Alert R blood culture bottles remained negative. Bartonella spp. structures within erythrocytes were confirmed by electron microscopy. The viability of B. henselae was demonstrated after a storage period of RBC units. These data reinforce the possibility of infection by transfusion of blood units collected from asymptomatic blood donors.

Detection of human herpesvirus 6 in patients with oral chronic graft-vs-host disease following allogeneic progenitor cell transplantation.

INTRODUCTION: Chronic graft-vs-host disease (cGVHD) is a major cause of morbidity in long-term survivors of allogeneic hematopoietic progenitor cell transplantation. Herpesviruses are involved in the occurrence and progression of various oral diseases. AIM: The aim of this study was to investigate the role of human herpesvirus 6 (HHV6) in patients with oral manifestations of cGVHD. MATERIALS AND METHODS: Peripheral blood and oral fluids (whole saliva, gingival crevicular fluid and parotid gland saliva) from 19 cGVHD patients, and 28 blood donors were examined for HHV6. Oral tissue samples were collected from 12 cGVHD patients and 12 healthy individuals. Nested polymerase chain reaction was employed to identify the HHV6. RESULTS AND CONCLUSION: The virus was detected in whole saliva in 13 cGVHD patients (68%) and in 19 blood donors (67%). HHV6 was not identified in any of the gingival crevicular fluid and parotid gland saliva samples in cGVHD patients. In the control group 14.3% of both, four gingival crevicular fluid and four parotid gland saliva samples were positive. Two oral tissue samples of cGVHD patients were positive for HHV6. These results indicate that patients with oral manifestations of cGVHD and healthy individuals present high and similar incidence of HHV6 in blood and oral fluids. These data do not support the importance of HHV6 in oral lesions of cGVHD.

Association of ABO gene mutations resulting in a rare B subgroup.

BACKGROUND AND OBJECTIVES: B subgroups are rare and the genetic analysis reported to date has been limited. MATERIALS AND METHODS: Serological and molecular investigations were performed in blood from a B-subgroup donor. RESULTS: Red cells did not react with anti-B and anti-AB reagents. However, cells absorbed anti-B. Red cells presented positive reactions with anti-H, and saliva secreted H substance. The molecular study demonstrated a B allele with the substitutions 467C>T, 646T>A, 681G>A, 771C>T, 796C>A, 803G>C, 829G>A and an O allele with the sequence of O02. CONCLUSIONS: It is probable that the presence in exon 7 of some of the O02 substitutions could have weakened the enzymatic activity of the encoded B transferase.

Elastic properties of stored red blood cells from sickle trait donor units.

BACKGROUND AND OBJECTIVES: Red blood cells (RBCs) from patients with sickle cell disease present reduced deformability. The aim of this study was to analyse the elasticity of stored RBCs from patients with the sickle cell trait (AS). MATERIALS AND METHODS: The cell elasticity was studied, using laser optical tweezers, on storage days 1, 14, 21, 28 and 35. RESULTS: The elasticity of RBC from AS units stored for 1, 14 and 21 days was significantly greater compared with that of control RBC cells stored for the same time-period. More than 30% of the cells from AS units stored for 28 or 35 days were very rigid and escaped from the optical trap. CONCLUSIONS: RBCs became rigid during storage, suggesting that haemoglobin S might compromise the cell elasticity.

Severe immune haemolysis in a group A recipient of a group O red blood cell unit.

Haemolysis caused by passive ABO antibodies is a rare transfusional complication. We report a case of severe haemolytic reaction in a 38-year-old man (blood group A) with lymphoma who had received one red blood cell (RBC) unit group O. After transfusion of 270 mL, the patient experienced fever, dyspnoea, chills and back pain. On the following morning he was icteric and pale. Haptoglobin was inferior to 5.8 mgdL(-1), haemoglobin was not increased and lactate dehydrogenase was elevated. Haemolysis was evident on observation of the patient's post-transfusion samples. The recipient's red cells developed a positive direct antiglobulin test and Lui elution showed anti-A coated the cells. Fresh donor serum had an anti-A titre of 1024, which was not reduced by treating the serum with dithiothreitol. Donor isoagglutinin screening has been determined by microplate automated analyser and showed titre higher than 100. Physicians should be aware of the risk of haemolysis associated with ABO-passive antibodies. There is generally no agreement justifying the isoagglutinin investigation prior to transfusion. However, automated quantitative isoagglutinin determination could be part of the modern donor testing process, mainly in blood banks where identical ABO group units (platelets or phenotyped RBCs) are not available owing to limited supply.

Optical tweezers for measuring red blood cell elasticity: application to the study of drug response in sickle cell disease.

The deformability of erythrocytes is a critical determinant of blood flow in microcirculation. By capturing red blood cells (RBC) with optical tweezers and dragging them through a viscous fluid we were able to measure their overall elasticity. We measured, and compared, the RBC deformability of 15 homozygous patients (HbSS) including five patients taking hydroxyurea (HU) for at least 6 months (HbSS/HU), 10 subjects with sickle cell trait (HbAS) and 35 normal controls. Our results showed that the RBC deformability was significantly lower in haemoglobin S (HbS) subjects (HbSS and HbAS), except for HbSS/HU cells, whose deformability was similar to the normal controls. Our data showed that the laser optical tweezers technique is able to detect differences in HbS RBC from subjects taking HU, and to differentiate RBC from normal controls and HbAS, indicating that this is a very sensitive method and can be applied for detection of drug-response in sickle cell disease.

ABO blood group in Amerindians from Brazilian Amazon.

BACKGROUND: The Parakanã is a group of Indians with cultural similarities to the extinct Tupi group. They are an isolated native population from East Brazilian Amazon. A number of different O alleles have been found at the blood group ABO locus in populations of several ethnic origins (Caucasians, Blacks, Amerindians). AIM: The present study describes the ABO blood group polymorphism gene of the Parakanã Indians. The Amerindian group was carefully selected for racial background. SUBJECT AND METHODS: The blood group polymorphism was analysed in genomic DNA from 62 Parakanã Indians. We determined the 261G deletion, the T646A and C771T mutations described in O(1variant) and the G542A substitution, using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism). RESULTS: All Amerindians studied were homozygous for the 261G deletion. The frequencies of the T646A and C771T mutations in Parakanãs (0.65) were lower than that observed in Kayapo, Yanomama and Arara Indians (0.91) (chi (2) = 18.24; p-v < 0.001. The G542A substitution in Parakanãs was also lower (0.22) than in other tribes (0.42) (chi(2) = 9.73; p-v = 0.001). CONCLUSIONS: The different O alleles including the G542A mutation are not distributed homogeneously among all Amazonian Amerindians. Our results are in agreement with other genetic markers studied previously in Parakanã Indians, whose distinct genetic pattern differs from Europeans and even from other Amerindians.

Elastic properties of irradiated RBCs measured by optical tweezers.

BACKGROUND: Gamma irradiation of RBCs results in the production of reactive oxygen capable of initiating the process of membrane lipid peroxidation and accelerates the leakage of potassium ions from RBCs, resulting in an increase of internal viscosity. STUDY DESIGN AND METHODS: The elastic properties of irradiated and stored RBC units were studied using laser optical tweezers. The laser trapped the cell and the membrane elasticity was analyzed, measuring the cell deformation in six different drag velocities. Five RBC units were split into two portions. One portion received a gamma irradiation dose of 25 Gy, and the second one was used as a control and was not irradiated. All units were stored (4 degrees C), and the elasticity was examined on Days 1, 14, 21, and 28. RESULTS: Elastic properties (mu) from irradiated RBCs stored for 21 and 28 days were significantly affected compared with control cells (21 days: control, 0.3 +/- 0.03 x 10(-3); irradiated, 3.5 +/- 1.3 x 10(-3) dyn/cm; p < 0.001; and 28 days: control, 0.5 +/- 0.09 x 10(-3); irradiated, 14 +/- 3.2 x 10(-3) dyn/cm; p < 0.001). CONCLUSION: The sensitivity of the laser optical tweezers method showed that there is no significant change in elasticity over time for up to 14 days of storage, regardless of whether the unit was irradiated or not. However, beyond 21 days of storage, irradiated units demonstrate decreased elasticity.

Molecular heterogeneity of the A3 subgroup.

The molecular characterization of the subgroup A3 remains unclear. Four unrelated A3 blood donors were studied. Family studies were possible in three of them. The A3 subgroup was defined by immunohaematological evaluation with four different commercially available serums. Exons VI and VII of the ABO gene, responsible for 91% of the catalytic active part of the glycosyltransferase, were amplified and subjected to direct sequencing. The results in all samples showed heterozygosity for the G261 deletion. In the A3 allele, the following associations were found: C467T mutation and 1060C deletion in one A3 blood donor and in another G829A and 1060C. In one case, only the 1060C deletion was demonstrated in the A3 allele. One blood donor presented the T646A and the G829A mutations in homozygosity. It was concluded that the A3 blood group is very heterogeneous at the molecular level.