Relevance of blood groups in transfusion of sickle cell disease patients

Blood groups are clinically significant in sickle cell disease (SCD) as transfusion remains a key treatment in this pathology. The occurrence of a delayed haemolytic transfusion reaction (DHTR) is not rare and is a life-threatening event. The main cause of DHTR is the production of alloantibodies against red blood cell antigens. The high rate of alloimmunization in SCD patients is mainly due to the differences of red blood groups between patients of African descent, and the frequently Caucasian donors. From an immuno-haematological point of view, DHTR in SCD patients has specific features: classical antibodies known to be haemolytic can be encountered, but otherwise non significant antibodies, autoantibodies and antibodies related to partial and rare blood groups are also frequently found in individuals of African descent. In some cases, there are no detectable antibodies. As alloimmunization remains the main cause of DHTR, it is extremely important to promote blood donation by individuals of African ancestry to make appropriate blood available.     

Development of a method for the determination of zopiclone in whole blood

Mass spectrometry in both electron-impact and positive chemical ionisation modes has been used to elucidate the structures of the decomposition products of zopiclone after gas chromatography. A high-performance liquid chromatographic method has been developed for the determination of zopiclone in whole blood. After selective extraction (butyl chloride) the extracts are chromatographed on Spherisorb ODS-5 (5 μm) using dibasic ammonium phosphate—acetonitrile (40:60). The zopiclone is measured by ultraviolet detection with a limit of quantitation of 4 ng/ml. This method has been successfully applied to the determination of zopiclone in post-mortem blood. Zopiclone levels found in five post-mortem cases are presented.     

All clinically-relevant blood components transmit prion disease following a single blood transfusion: a sheep model of vCJD

Variant CJD (vCJD) is an incurable, infectious human disease, likely arising from the consumption of BSE-contaminated meat products. Whilst the epidemic appears to be waning, there is much concern that vCJD infection may be perpetuated in humans by the transfusion of contaminated blood products. Since 2004, several cases of transfusion-associated vCJD transmission have been reported and linked to blood collected from pre-clinically affected donors. Using an animal model in which the disease manifested resembles that of humans affected with vCJD, we examined which blood components used in human medicine are likely to pose the greatest risk of transmitting vCJD via transfusion. We collected two full units of blood from BSE-infected donor animals during the pre-clinical phase of infection. Using methods employed by transfusion services we prepared red cell concentrates, plasma and platelets units (including leucoreduced equivalents). Following transfusion, we showed that all components contain sufficient levels of infectivity to cause disease following only a single transfusion and also that leucoreduction did not prevent disease transmission. These data suggest that all blood components are vectors for prion disease transmission, and highlight the importance of multiple control measures to minimise the risk of human to human transmission of vCJD by blood transfusion.     

Cytokine secretion depends on Galalpha(1,3)Gal expression in a pig-to-human whole blood model

Transplants from alpha1,3-galactosyltransferase (Gal) gene-knockout pigs to nonhuman primates are largely protected from hyperacute but not acute humoral xenograft rejection. The present study investigates the role of Gal in cytokine responses using a novel pig-to-human whole blood in vitro model, developed for species-specific analysis of porcine and human cytokines. Porcine (n = 7) and human (n = 27) cytokines were measured using ELISA or multiplex technology, respectively. Porcine aortic endothelial cells from control (Gal(+/+)) and Gal-deficient (Gal(-/-)) pigs were incubated with human lepirudin anticoagulated whole blood from healthy donors. E-selectin expression was measured by flow cytometry. The C3 inhibitor compstatin and a C5aR antagonist were used to study the role of complement. Cytokine species specificity was documented, enabling detection of 2 of 7 porcine cytokines and 13 of 27 human cytokines in one single sample. Gal(+/+) porcine aortic endothelial cells incubated with human whole blood showed a marked complement C5b-9 dependent up-regulation of E-selectin and secretion of porcine IL-6 and IL-8. In contrast, Gal(-/-) cells responded with E-selectin and cytokine expression which was so weak that the role of complement could not be determined. Human IL-6, IL-8, IFN-gamma, MIP-1alpha, MIP-1beta, eotaxin, and RANTES were detected in the Gal(+/+) system, but virtually no responses were seen in the Gal(-/-) system (p = 0.03). The increase in human cytokine release was largely complement dependent and, in contrast to the porcine response, mediated through C5a. Species-specific analysis of cytokine release revealed a marked, complement-dependent response when Gal(+/+) pig cells were incubated with human whole blood, compared with Gal(-/-) cells which induced virtually no cytokine release.     

Micro-buffy coats of whole blood: a method for the electron microscopic study of mononuclear cells

A method for the electron microscopic study of human peripheral lymphocytes by which very small buffy coats are obtained through centrifugation of heparinized whole blood in glass or plastic microhematocrit tubes is presented. This method is time saving and efficient, yielding well preserved material and a comparatively large number of mononuclear cells (mainly lymphocytes) in each thin section.     

Binding features of diacetylmorphine (heroin) in whole blood and in blood fractions.

Binding features of heroin in whole blood and in blood fractions were delineated by measuring the selective spin-lattice relaxation rates of heroin protons in physiologic conditions. Interaction with some receptor located in the whole human blood or in the human plasma was detected and the apparent binding constant calculated (K = 39 mol-1 dm3). Inferences about molecular dynamics of the bound heroin could be also gained.     

Peptidylpropyl isomerase B (PPIB): a suitable reference gene for mRNA quantification in peripheral whole blood

Quantitative real-time RT-PCR is a very powerful technique for measuring gene expression at the mRNA level. In order to compare mRNA expression in different experimental or clinical conditions, expression of a target gene has to be normalized to an appropriate internal standard, which is generally a housekeeping gene. In our study, we have tested several housekeeping genes in peripheral whole blood of healthy volunteers and patients suffering from inflammatory diseases. A first analysis of 91 samples illustrated that the mRNA expression of peptidylpropyl isomerase B (PPIB) encoding for cyclophilin B protein, is more stable than beta actin and glyceraldehyde-3-phosphate dehydrogenase, which are both commonly selected as internal standard. Among the three genes tested, beta actin displayed the highest inter-sample variation of expression. The constancy of PPIB mRNA expression was further confirmed by 214 additional samples. In conclusion, we showed that PPIB, in contrast to beta actin and glyceraldehyde-3-phosphate dehydrogenase, is a suitable housekeeping gene in human peripheral blood.     

Influence of the storage time and the method of stimulation on whole blood chemiluminescence

The ultra‐weak light, chemiluminescence (CL), of stimulated leukocytes is a well‐known phenomenon. Parameters of this CL are modified by many factors including laboratory procedures. The order of stimulation and enhancement (two possibilities) and two concentrations of luminol create four types of procedure, which were accomplished in five sample storage ‘time points’. We received the strongest signals of CL using higher concentrations of luminol (and DMSO), but only when stimulation (FMLP) was used before enhancement (luminol); luminol used before FMLP strongly inhibited CL. For lower luminol concentration (and DMSO), the order of stimulation and enhancement was of no importance. There were comparable but weaker signals of CL in this case. We received stronger signals with storage time for all procedures. It may be dependent on the priming of phagocytes by releasing cell factors. Stimulation (FMLP) before enhancement (luminol) eliminates the inhibitory effect of DMSO on CL. Copyright © 2002 John Wiley & Sons, Ltd.     

Regulation of immune activity by mild (fever-range) whole body hyperthermia: effects on epidermal Langerhans cells

Inflammation of the skin and systemic fever, both of which occur with injury or infection, include a hyperthermic component that many believe constitutes a physiological stress. Such increases in local or systemic body temperature may also have a regulatory effect on immune function. Langerhans cells (LCs), the dendritic cells of the skin, continuously monitor the extracellular matrix of the skin by taking up particles and microbes that they then carry to draining lymph nodes for presentation to T lymphocytes. We hypothesize that the thermal element of inflammation and/or fever may help regulate the activation and migration of LCs out of the epidermis. To test this hypothesis, Balb/ c mice were exposed to a mild (39.8 degrees C +/- 0.2 degrees C), long-duration (6 hours) whole body hyperthermia (WBH) treatment, which mimics the thermal component of fever. The number of LCs and their morphology were analyzed at various time points up to 7 days after the initiation of WBH. The LCs of the ear epidermis were visualized using a fluorescein isothiocyanate-conjugated antibody specific for the major histocompatibility complex (MHC) class II molecule and confocal microscopy. Although MHC class II staining was diffuse on the surface of the LC body and dendritic extensions of both WBH and control samples, the WBH-treated LCs exhibited a more punctate morphology with fewer dendritic processes compared with control LCs. A significant decrease in the number of LCs was also observed 1 to 5 days after WBH treatment. Furthermore, in vitro heating of Balb/c ear skin cultures at 40 degrees C for 6 to 8 hours enhanced the numbers of viable LCs that migrated into the culture wells. These results suggest that WBH treatment stimulates epidermal LCs in the absence of foreign antigen.     

Up-regulation of blood arachidonate (20:4) levels in patients with chronic obstructive pulmonary disease

Context and objective: Plasma arachidonate (20:4) levels in patients with chronic obstructive pulmonary disease (COPD) were investigated.

Methods: Plasma was extracted and free fatty acids (FFAs) were separated using column chromatography and measured by fluorescence. Plasma 20:4 levels and its percentage relative to total FFA levels (%20:4) were measured in COPD (n = 18) and control (n = 20) subjects.

Results and conclusions: FFA levels were lower in COPD compared with normals. However, there was a significant increase in %20:4 levels in COPD patients (GOLD stage I/II 0.9 ± 0.4%; GOLD stage III/IV 1.1 ± 0.1%) compared with control subjects (0.6 ± 0.1, p < 0.05). %20:4 is a potential biomarker for COPD.     

Nucleoli of lymphocytes in the peripheral blood of patients with periodontal disease (preliminary report)

A group of 104 patients with various grades of severity of the periodontal disease and a control group of healthy volunteers were examined. On the basis of an examination of the nucleoli, the activity of the RNA synthesis in the lymphocytes of peripheral blood was established. Patients with a very advanced periodontal disease showed a statistically highly significant increase in the ratio of active and reversibly inactivated lymphocytes. Thus, the significance of immune factors in the development of the periodontal disease was confirmed.     

Molybdenum in human whole blood of adult residents of the Merida State (Venezuela)

The concentration of molybdenum was measured in whole blood samples of 418 (244 males and 174 females) apparently normal donors ranging in age from 18 to 27-years old and living in nine different locations in the Mérida State (Venezuela). The geometric mean concentration of molybdenum of 418 subjects was of 2.66+/-0.66 microgL(-1) (range: 1.20-4.80 microgL(-1)). The levels of molybdenum in whole blood samples found in this work were of 2.57+/-0.52 and 2.54+/-0.51 (range: 1.20-4.80 and 1.40-4.20) microgL(-1) for males and females, respectively. The data of the content molybdenum in whole blood had no statistical correlation with age, sex or height above the sea level of the sampling sites. However, there was a tendency to decrease the levels of the element in those sampling sites located in highlands (> or = 1900 m above the sea level). This variability may be due to the source of molybdenum from the soil to the food chain that has affected its levels in donors from these areas under study. The results of this study are compared with values previously reported for subjects studied in other populations.