HDL associates with insulin resistance and beta-cell dysfunction in South Asian families at risk of type 2 diabetes.

OBJECTIVE: Dyslipidemia precedes type 2 diabetes (T2D) and worsens with increasing glucose intolerance. First degree relatives of T2D patients have an increased risk to develop dyslipidemia and glucose intolerance. The aim of the present study was to assess the relation between the development of dyslipidemia and glucose intolerance in first-degree relatives of T2D patients. RESEARCH DESIGN AND METHODS: Fasting lipoprotein profiles were determined by density gradient ultracentrifugation in T2D patients and their first-degree relatives (42 Caucasians and 33 South Asians), and in 29 normoglycemic controls from non-T2D families. Glucose tolerance, insulin sensitivity index (ISI) and insulin disposition index (DI) were assessed by an extended, frequently sampled oral glucose tolerance test (OGTT), and fractional insulin synthesis rate (FSR) was measured by 13C-leucine enrichment in urinary C-peptide during the OGTT. RESULTS: Of the first-degree relatives, 40, 16 and 19 had NGT, prediabetes and T2D, respectively. NGT family members had lower plasma HDL-cholesterol (HDLC) (1.34 ± 0.07 vs 1.58 ± 0.06 mmol/L; p = 0.015), HDL2-C (0.41 ± 0.05 vs 0.57 ± 0.05 mmol/L; p = 0.021) and HDL3-C (0.62 ± 0.03 vs 0.72 ± 0.02 mmol/L; p = 0.043) than controls. HDL2-C levels tended to decrease with increasing glucose intolerance state. In South Asians, buoyant LDL-C levels decreased with increasing glucose intolerance state (p = 0.006). In South Asian families, HDL-C correlated with both ISI and DI (ß 0.42; p = 0.04 and ß 0.53; p = 0.01, respectively), whereas HDL2-C and HDL3-C levels correlated with DI (ß 0.64; p = 0.002 and ß 0.57; p = 0.005, respectively). HDL2-C and plasma triglyceride correlated with FSR (ß 0.48; p = 0.033 and ß -0.50; p = 0.029, respectively). CONCLUSIONS: Low HDL2-C and HDL3-C levels are present in NGT first-degree relatives of T2D patients, and HDL2-C tend to decrease further with increasing glucose intolerance. In South Asian families HDL2-C and HDL3-C levels linked predominantly to deteriorating beta cell function.

Breakfast partly restores the anti-inflammatory function of high-density lipoproteins from patients with type 2 diabetes mellitus.

Background and aims: High-density lipoproteins (HDL) of patients with type 2 diabetes mellitus (T2DM) have impaired anti-inflammatory activities. The anti-inflammatory activity of HDL has been determined ex vivo after isolation by different methods from blood mostly obtained after overnight fasting. We first determined the effect of the HDL isolation method, and subsequently the effect of food intake on the anti-inflammatory function of HDL from T2DM patients. Methods: Blood was collected from healthy controls and T2DM patients after an overnight fast, and from T2DM patients 3 h after breakfast (n = 17 each). HDL was isolated by a two-step density gradient ultracentrifugation in iodixanol (HDLDGUC2), by sequential salt density flotation (HDLSEQ) or by PEG precipitation (HDLPEG). The anti-inflammatory function of HDL was determined by the reduction of the TNFα-induced expression of VCAM-1 in human coronary artery endothelial cells (HCAEC) and retinal endothelial cells (REC). Results: HDL isolated by the three different methods from healthy controls inhibited TNFα-induced VCAM-1 expression in HCAEC. With apoA-I at 0.7 µM, HDLDGUC2 and HDLSEQ were similarly effective (16% versus 14% reduction; n = 3; p > 0.05) but less effective than HDLPEG (28%, p < 0.05). Since ultracentrifugation removes most of the unbound plasma proteins, we used HDLDGUC2 for further experiments. With apoA-I at 3.2 µM, HDL from fasting healthy controls and T2DM patients reduced TNFα-induced VCAM-1 expression in HCAEC by 58 ± 13% and 51 ± 20%, respectively (p = 0.35), and in REC by 42 ± 13% and 25 ± 18%, respectively (p < 0.05). Compared to preprandial HDL, postprandial HDL from T2DM patients reduced VCAM-1 expression by 56 ± 16% (paired test: p < 0.001) in HCAEC and by 34 ± 13% (paired test: p < 0.05) in REC. Conclusions: The ex vivo anti-inflammatory activity of HDL is affected by the HDL isolation method. Two-step ultracentrifugation in an iodixanol gradient is a suitable method for HDL isolation when testing HDL anti-inflammatory function. The anti-inflammatory activity of HDL from overnight fasted T2DM patients is significantly impaired in REC but not in HCAEC. The anti-inflammatory function of HDL is partly restored by food intake.

Lomitapide affects HDL composition and function.

BACKGROUND: Lomitapide reduces low-density lipoprotein-cholesterol (LDL-C) but also high-density lipoprotein-cholesterol (HDL-C) levels. The latter may reduce the clinical efficacy of lomitapide. We investigated the effect of lomitapide on HDL-C levels and on cholesterol efflux capacity (CEC) of HDL in patients with homozygous familial hypercholesterolemia (HoFH). METHODS AND RESULTS: Four HoFH patients were treated with increasing dosages of lomitapide. Lomitapide decreased LDL-C (range -34 to -89%). Total HDL-C levels decreased (range -16 to -34%) with a shift to buoyant HDL. ABCA1-mediated CEC decreased in all patients (range -39 to -99%). The changes of total, ABCG1- and SR-BI-mediated CEC were less consistent. CONCLUSION: Lomitapide decreased LDL-C and HDL-C levels. Our report raises the hypothesis that the anti-atherogenic potential of HDL seems to be unaffected as total CEC did not seem to change consistently. Combined with the reduction of atherogenic lipoproteins, the net effect of lomitapide appears to be beneficial in HoFH patients.

Diet-induced weight loss and markers of endothelial dysfunction and inflammation in treated patients with type 2 diabetes.

BACKGROUND & AIMS: Overweight and obesity increase cardiovascular mortality in patients with type 2 diabetes (T2D). In a recent trial, however, diet-induced weight loss did not reduce the cardiovascular risk of patients with T2D, possibly due to the parallel intensive medical treatment. We investigated the effect of diet-induced weight loss on cardiovascular risk factors in overweight and obese patients with T2D, and whether this effect was influenced by the use of statins, ACE inhibitors, metformin and duration of T2D. METHODS: Patients with T2D and BMI >27 were subjected to an energy-restricted diet during 4 months. Before and after intervention, plasma levels of sICAM-1, sVCAM-1, hsCRP, vWF and classical biomarkers were measured. The association of the change in biomarker levels with medication use and T2D history, corrected for age, sex and change in insulin dose, was tested by matched linear regression analyses. RESULTS: In 131 patients, the diet resulted in weight loss of 10.2 kg (95%CI 9.2, 11.3; p < 0.001), improved median levels of HbA1c (-7.0 mmol/mol (95%CI -8.5, -5.0); p < 0.001), LDL cholesterol (-0.2 mmol/L (95%CI -0.4, -0.1); p < 0.001), sICAM-1 (-22.4 ng/mL (95%CI -37.1, -8.7); p = 0.001), vWF (-3.9 IU/mL (95%CI -6.4, -1.4); p = 0.003) and hs-CRP (-0.6 mg/L (95%CI -1.2, -0.2); p = 0.007), but did not affect sVCAM-1 levels (1.6 ng/mL (95%CI -41.5, 48.6); p = 0.949). Duration of T2D and medical treatment were not associated with these effects, except for an association between statin use and change in sVCAM-1, where statin users improved more. CONCLUSION: Diet-induced weight loss reduced the levels of biomarkers of endothelial dysfunction and inflammation in overweight and obese patients with T2D independently of medication use and T2D duration. Even on intensive medical drug treatment as well as after a long history of T2D, patients may still profit from diet-induced weight reduction.

Lipoprotein(a) levels are associated with aortic valve calcification in asymptomatic patients with familial hypercholesterolaemia.

OBJECTIVES: Lipoprotein(a) [Lp(a)] is an independent risk factor for aortic valve stenosis and aortic valve calcification (AVC) in the general population. In this study, we determined the association between AVC and both plasma Lp(a) levels and apolipoprotein(a) [apo(a)] kringle IV repeat polymorphisms in asymptomatic statin-treated patients with heterozygous familial hypercholesterolaemia (FH). METHODS: A total of 129 asymptomatic heterozygous FH patients (age 40-69 years) were included in this study. AVC was detected using computed tomography scanning. Lp(a) concentration and apo(a) kringle IV repeat number were measured using immunoturbidimetry and immunoblotting, respectively. Univariate and multivariate logistic regression were used to assess the association between Lp(a) concentration and the presence of AVC. RESULTS: Aortic valve calcification was present in 38.2% of patients, including three with extensive AVC (>400 Agatston units). Lp(a) concentration was significantly correlated with gender, number of apo(a) kringle IV repeats and the presence and severity of AVC, but not with coronary artery calcification (CAC). AVC was significantly associated with plasma Lp(a) level, age, body mass index, blood pressure, duration of statin use, cholesterol-year score and CAC score. After adjustment for all significant covariables, plasma Lp(a) concentration remained a significant predictor of AVC, with an odds ratio per 10-mg dL(-1) increase in Lp(a) concentration of 1.11 (95% confidence interval 1.01-1.20, P = 0.03). CONCLUSION: In asymptomatic statin-treated FH patients, plasma Lp(a) concentration is an independent risk indicator for AVC.

No difference in glycosphingolipid metabolism and mitochondrial function in glucocorticoid-induced insulin resistance in healthy men.

OBJECTIVE: Glucocorticoids (GCs) are well known to induce insulin resistance; however, mechanisms that cause the impairement of the insulin signaling pathway have not yet been identified. In this study we measured whether GC-induced insulin resistance in humans is related to changes in muscle ceramide, GM3, and muscle mitochondrial function. METHODS: In a randomized, placebo-controlled, double-blind, dose-response intervention study, 32 healthy males (aged 22 ± 3 years; body mass index 22.4 ± 1.7 kg/m(-2)) were allocated to prednisolone (PRED) 7.5 mg once daily (n = 12), PRED 30 mg once daily (n = 12), or placebo (n = 8) for 2 weeks using block randomization. Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp before and after treatment. Muscle biopsies were performed to measure ceramide, monosialodihexosylganglioside (GM3), and mitochondrial function. RESULTS: Peripheral insulin sensitivity was dose dependently decreased after the PRED treatment. Muscle ceramide and GM3 concentration and mitochondrial function were not altered by 2 weeks of PRED treatment. CONCLUSION: Short-term GC treatment dose dependently impaired whole-body insulin sensitivity in healthy males, without concomitant changes in muscle ceramide, GM3, or mitochondrial function. These findings suggest that other mechanisms play a role in GC-related impairment of insulin sensitivity.

Glucose increases hepatic lipase expression in HepG2 liver cells through upregulation of upstream stimulatory factors 1 and 2.

AIMS/HYPOTHESIS: Elevated hepatic lipase (HL, also known as LIPC) expression is a key factor in the development of the atherogenic lipid profile in type 2 diabetes and insulin resistance. Recently, genetic screens revealed a possible association of type 2 diabetes and familial combined hyperlipidaemia with the USF1 gene. Therefore, we investigated the role of upstream stimulatory factors (USFs) in the regulation of HL. METHODS: Levels of USF1, USF2 and HL were measured in HepG2 cells cultured in normal- or high-glucose medium (4.5 and 22.5 mmol/l, respectively) and in livers of streptozotocin-treated rats. RESULTS: Nuclear extracts of cells cultured in high glucose contained 2.5 +/- 0.5-fold more USF1 and 1.4 +/- 0.2-fold more USF2 protein than cells cultured in normal glucose (mean +/- SD, n = 3). This coincided with higher DNA binding of nuclear proteins to the USF consensus DNA binding site. Secretion of HL (2.9 +/- 0.5-fold), abundance of HL mRNA (1.5 +/- 0.2-fold) and HL (-685/+13) promoter activity (1.8 +/- 0.3-fold) increased in parallel. In chromatin immunoprecipitation assays, the proximal HL promoter region was immunoprecipitated with anti-USF1 and anti-USF2 antibodies. Co-transfection with USF1 or USF2 cDNA stimulated HL promoter activity 6- to 16-fold. USF and glucose responsiveness were significantly reduced by removal of the -310E-box from the HL promoter. Silencing of the USF1 gene by RNA interference reduced glucose responsiveness of the HL (-685/+13) promoter region by 50%. The hyperglycaemia in streptozotocin-treated rats was associated with similar increases in USF abundance in rat liver nuclei, but not with increased binding of USF to the rat Hl promoter region. CONCLUSIONS/INTERPRETATION: Glucose increases HL expression in HepG2 cells via elevation of USF1 and USF2. This mechanism may contribute to the development of the dyslipidaemia that is typical of type 2 diabetes.

Identification by a differential proteomic approach of the induced stress and redox proteins by resveratrol in the normal and diabetic rat heart.

A recent study showed cardioprotective effects of resveratrol on the diabetic heart. The present study sought to compare the protein profiles of the normal versus diabetic hearts after resveratrol treatment using differential proteomic analysis. Rats were randomly divided into two groups: control and diabetic. Both groups of rats were fed resveratrol (2.5 mg/kg/day) for 7 days, and then the rats were sacrificed, hearts were isolated and cytoplasmic fraction from left ventricular tissue was collected to carry out proteomic profiling as well as immunoblotting. Compared to normal hearts, diabetic hearts show increased myocardial infarct size and cardiomy-ocyte apoptosis upon ex vivo global ischaemia of 30 min. followed by 2 hrs of reperfusion. Resveratrol reduced infarct size and apop-totic cell death for both the groups, but the extent of infarct size and apoptosis remained higher for the diabetic group compared to the normal group. The left ventricular cytoplasmic proteins were analysed by 2D-DIGE and differentially displayed bands were further analysed by nano Liquid Chromatography-Mass Spectroscopy (LC-MS/MS). The results showed differential regulation of normal versus diabetic hearts treated with resveratrol of many proteins related to energy metabolism of which several were identified as mitochondrial proteins. Of particular interest is the increased expression of several chaperone proteins and oxidative stress and redox proteins in the diabetic group including Hsc70, HSPp6, GRP75, peroxiredoxin (Prdx)-1 and Prdx-3 whose expression was reversed by resveratrol. Western blot analysis was performed to validate the up- or down-regulation of these stress proteins. The results indicate the differential regulation by resveratrol of stress proteins in diabetic versus normal hearts, which may explain in part the beneficial effects of resveratrol in diabetic induced cardiovascular complications.

Mitochondria in neutrophil apoptosis.

Central in the regulation of the short life span of neutrophils are their mitochondria. These organelles hardly contribute to the energy status of neutrophils but play a vital role in the apoptotic process. Not only do the mitochondria contain cytotoxic proteins that are released during apoptosis and contribute to caspase activation, but they also act as sensors of the metabolic and redox state of the cell and as scavengers of free Ca2+. The balance of the expression and activity of the proapoptotic and antiapoptotic members of the Bcl-2 family of proteins determines the life span of neutrophils, because these proteins are essential for the formation of a permeability transition pore in the mitochondria and also seem to control the release of Ca2+ from the endoplasmic reticulum and thereby mitochondrial energy metabolism.

Prolonged storage of red blood cells affects aminophospholipid translocase activity.

BACKGROUND AND OBJECTIVES: Loss of phospholipid asymmetry in the membrane of red blood cells (RBC) results in exposure of phosphatidylserine (PS) and to subsequent removal from the circulation. In this study, we investigated the effect of long-term storage of RBCs on two activities affecting phospholipid asymmetry: the ATP-dependent aminophospholipid translocase (or flippase, transporting PS from the outer to the inner leaflet) and phospholipid scrambling (which will move PS from the inner to the outer leaflet). MATERIALS AND METHODS: Standard leukodepleted RBC concentrates were stored in saline-adenine-glucose-mannitol (SAGM) at 4 degrees C for up to 7 weeks. PS exposure was determined by measurement of AnnexinV-FITC binding to the cells, flippase activity by measurement of the inward translocation of NBD-labelled PS. Scrambling activity was determined by following the inward translocation of fluorescent NBD-phosphatidylcholine. In parallel, intracellular ATP levels were determined. RESULTS: PS exposure amounted to only 1.5 +/- 0.3% positive cells (n = 8) after 5 weeks of storage, which slightly increased to 3.5 +/- 0.7% (n = 8) after 7 weeks of storage. Flippase activity started to decrease after 21 days of storage and reached 81 +/- 5% of the control value after 5 weeks of storage (n = 6) and 59 +/- 6% (n = 6) after 7 weeks. Also in RBC obtained by apheresis, flippase activity decreased upon storage. Scrambling activity remained virtually absent during storage, explaining the low PS exposure despite the decrease in flippase activity. Rejuvenation of RBC after 7 weeks to increase ATP levels only partially restored flippase activity, but in combination with a correction of the intracellular pH to that of fresh cells, almost complete restoration was achieved. The decrease in flippase activity after prolonged storage did make the RBCs more prone to PS exposure after activation of phospholipid scrambling. CONCLUSION: This study shows that, although PS exposure remains low, prolonged storage does compromise the RBC membrane by affecting flippase activity. When the metabolic changes induced by storage are corrected, flippase activity can be restored.

Differential sensitivities of pathogens in red cell concentrates to Tri-P(4)-photoinactivation.

BACKGROUND AND OBJECTIVES: Photodynamic treatment (PDT) with the cationic porphyrin, mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin chloride [Tri-P(4)], has previously been shown to be effective at inactivating vesicle stomatitis virus (VSV) in red cell concentrates (RCC) with limited damage to red blood cells (RBC). The aim of this study was to determine the pathogen-inactivating capacity of PDT with Tri-P(4) for a broader range of pathogens and to establish the associated effect on in vitro RBC quality. MATERIALS AND METHODS: A series of viruses and bacteria was spiked into 60% RCC. Pathogen inactivation was determined after PDT with 25 microm Tri-P(4) and red light up to 360 kJ/m2. Human immunodeficiency virus (HIV)-infected cells were evaluated for cell death induction, and RCC were analysed for the induction of haemolysis and ATP content. RESULTS: For the lipid-enveloped viruses bovine viral diarrhoea virus, HIV and pseudorabies virus, and for the Gram positive bacterium, Staphylococcus aureus, and the Gram-negative bacteria, Pseudomonas aeruginosa and Yersinia enterolitica, inactivation of > or = 5 log10 was measured after 60 min of PDT with Tri-P(4). The required treatment time to achieve this level of inactivation was four times longer than required for VSV. For cell-associated HIV, only 1.7 log10 of inactivation was found, despite clear induction of cell death of HIV-infected cells. The non-enveloped virus, canine parvovirus, was completely resistant to the treatment. PDT of RCC with Tri-P(4) for 60 min, and subsequent storage in AS-3, resulted in 4% haemolysis after 35 days of storage. The ATP content of untreated and treated RBC declined with similar kinetics during storage. CONCLUSION: PDT of RCC with Tri-P(4) for 60 min inactivates a wide range of pathogens, but not cell-associated HIV and a non-enveloped virus, and compromises RBC quality. This reduces the suitability of PDT with Tri-P(4) for red cell sterilization. Therefore, further improvements in the treatment procedures to potentiate pathogen inactivation and to preserve RBC integrity will be required to generate an effective treatment for sterilizing RCC.

The age of stored red blood cell concentrates at the time of transfusion.

In the past decade, studies suggesting a reduced oxygen delivery by stored red blood cell concentrates (RBCCs) have initiated a discussion about the use of fresh versus old blood. We determined whether old RBCCs represent a significant part of the total of RBCCs issued. The age of RBCCs at the time of transfusion was determined in 74 084 units during a 5-year period in the Academic Medical Center, a main Dutch University Hospital. The mean (+/-SD) storage time of the total number of transfused RBCC was 19.4 +/- 7.0 days, and 37% were older than 3 weeks. As more than one-third of the transfused RBCC units are stored for longer than 3 weeks, the research examining differences in oxygen delivery between fresh and stored RBCC is relevant for packed RBC transfusion practice.

The effect of storage time of human red cells on intestinal microcirculatory oxygenation in a rat isovolemic exchange model.

OBJECTIVE: To determine whether the storage time of human leukodepleted red blood cell concentrates compromises intestinal microvascular oxygen concentration oxygen (muPo(2)) during isovolemic exchange transfusion at low hematocrit. DESIGN: Prospective, randomized, controlled study. SETTING: University research institute laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Intestinal muPo(2) was determined by Pd-porphyrin phosphorescence life-time measurements. MEASUREMENTS AND MAIN RESULTS: Rats were brought near to a state of oxygen supply dependency by hemodilution with a pasteurized plasma protein solution to a hematocrit of 14.3 +/- 1.1% (n = 24). Subsequently, an isovolemic exchange transfusion with human leukodepleted red blood cells, stored for 2-6 days (fresh, n = 8), 2-3 wks (intermediate, n = 8), or 5-6 wks (old, n = 8), was performed to determine whether intestinal muPo(2) would be preserved. Immunologic reactions were avoided by washing the red blood cell concentrates three times before use. Isovolemic exchange with fresh and intermediate red blood cells maintained muPo(2) whereas old cells decreased muPo(2) with 26%. Subsequent transfusion with red blood cells (hematocrit approximately 60%) until reaching a hematocrit of 32.4 +/- 2.1 % (n = 24) increased intestinal muPo(2) in all three groups to the same extent between 28% and 32%. No changes in red blood cell deformability, as determined by a Laser-assisted Optical Rotational Cell Analyzer, could be demonstrated during 5 wks of storage. CONCLUSION: This study shows that at low hematocrit, the oxygen-delivering capacity of human red blood cells stored 5-6 wks is reduced compared with fresh cells and red blood cells stored for an intermediate period. Although red blood cells stored for 2-3 wks are completely devoid of 2,3-diphosphoglycerate, their oxygen-delivering capacity to the intestines was the same as fresh red blood cells. Our study showed that red blood cell deformability was preserved during storage, suggesting that other mechanisms may account for the observed decrease in oxygen delivery by red blood cells stored 2-3 wks.

Quality determinants of erythrocyte destined for transfusion.

An overview is given of a series of standard assays to evaluate the quality of red cell concentrates for transfusion. These are visual inspection, assessment of hemolysis, quantitation of 2,3-DPG and nucleotide levels (especially ATP) and evaluation of morphology. These parameters, relatively easy to measure, are main determinants of in vivo recovery after transfusion. In addition, some other assays are described, which should give more information about the function of red blood cells after transfusion. These assays include plasma-induced hemolysis, binding of annexin-V, deformability measurements and a rat model to judge oxygen delivery by human red blood cells (RBC). Especially in judging new protocols for the preparation of red cell products, involving e.g. improved additive solutions or pathogen inactivation methods, these quality parameters should not be compromised.

Generation of singlet oxygen induces phospholipid scrambling in human erythrocytes.

Maintenance of phospholipid asymmetry of the plasma membrane is essential for cells to prevent phagocytic removal or acceleration of coagulation. Photodynamic treatment (PDT), which relies on the generation of reactive oxygen species to achieve inactivation of pathogens, might be a promising approach in the future for decontamination of red blood cell concentrates. To investigate whether PDT affects phospholipid asymmetry, erythrocytes were illuminated in the presence of 1,9-dimethyl-methylene blue (DMMB) as photosensitizer and subsequently labeled with FITC-labeled annexin V. This treatment resulted in about 10% annexin V positive cells, indicating exposure of phosphatidylserine (PS). Treatment of erythrocytes with N-ethylmaleimide (NEM) prior to illumination, to inhibit inward translocation of PS via the aminophospholipid translocase, resulted in enhanced PS exposure, while treatment with H(2)O(2) (previously shown to inhibit phospholipid scrambling) greatly diminished PS exposure, indicating the induction of phospholipid scrambling by PDT. Only erythrocytes illuminated in the presence of DMMB showed translocation of NBD-phosphatidylcholine (NBD-PC), confirming scrambling induction. Double label experiments indicated that PS exposure does not occur without concurrent scrambling activity. Induction of scrambling was only moderately affected by Ca(2+) depletion of the cells. In contrast, scavengers of singlet oxygen were found to prevent phospholipid scrambling induced by PDT. The results of this study show that phospholipid scrambling is induced in human erythrocytes by exposure to singlet oxygen.

Comparison of various dimethylsulphoxide-containing solutions for cryopreservation of leucoreduced platelet concentrates.

BACKGROUND AND OBJECTIVES: Leucoreduced platelet concentrates (LR-PCs) can be stored at 20-24 degrees C for 5-7 days. When LR-PCs are cryopreserved they can be stored for several years. For cryopreservation to become applicable in blood-bank practice, an off-the-shelf cryoprotectant is needed that can be added to the LR-PC in a sterile manner. For this, we varied the composition of the cryopreservation medium and studied various parameters of cryopreserved LR-PCs for up to 24 h after thawing at room temperature. MATERIALS AND METHODS: LR-PCs in plasma or Composol were concentrated and divided into 2 units. To each unit, an equal part of 10% dimethylsulfoxide (DMSO) in plasma, Composol with or without 5% albumin, or GPO (pasteurized plasma-protein solution) was added. Freezing occurred at 1 degrees C/min and LR-PCs were placed in the vapour phase of nitrogen. LR-PCs were thawed at 37 degrees C and stored at room temperature. LR-PCs were tested for morphology, platelet recovery, swirling effect, and activation antigens at various time-points thereafter. RESULTS: LR-PCs in 100%, 65% and 50% plasma supplemented with Composol showed good morphology scores (>250), limited CD62P expression (<35%), low CD63 expression (<20%) and a swirling effect of about 2, at 24 h after thawing. At the same time-point, platelet recovery was >80% under all conditions and CD42b expression varied between 70 and 85%. Results of LR-PCs in 15% plasma and Composol, with or without plasma substitutes, were not acceptable at 24 h after thawing, i.e. the morphology score was <200 and the CD62P expression was >40%. CONCLUSIONS: A minimum of 50% plasma in the cryopreserved LR-PC is necessary to maintain an acceptable in vitro quality of platelets up to 24 h after thawing. Composol is a good candidate for using to prepare an off-the-shelf cryoprotectant.

Composition of the additive solution affects red blood cell integrity after photodynamic treatment.

BACKGROUND AND OBJECTIVES: Photodynamic treatment is a promising technique for pathogen inactivation of red blood cell concentrates. For protocol optimization, the influence of the composition of the storage solution on the integrity of phototreated red cells was studied. MATERIALS AND METHODS: Red blood cells were resuspended in the storage solutions SAG-M or AS-3 to a haematocrit (Hct) of 30%. After addition of the photosensitizer, 1,9-dimethylmethylene blue (DMMB) (25 microm), the suspensions were illuminated with red light, and potassium leakage and delayed haemolysis were determined. In some experiments, the cells were washed after illumination and resuspended in modified storage solutions. RESULTS: Illumination of red cells in the presence of DMMB resulted in an immediate, light-dose-dependent increase in potassium leakage. The illumination conditions used induced no detectable haemolysis immediately after photodynamic treatment. Potassium leakage was higher when the illumination was performed in AS-3. In contrast, delayed haemolysis, measured after overnight storage, was considerably lower when cells were stored in AS-3. This protection was mainly a result of the presence of citrate in AS-3. In addition, other impermeant solutes protected against haemolysis. CONCLUSIONS: The additive solution strongly influences the integrity of red cells after photodynamic treatment. Whereas the solution in which the cells are illuminated has a small effect on red cell integrity, the main influence of the additive solution is during post-treatment storage. Red cell integrity is best maintained when illumination is performed in SAG-M followed by storage in AS-3. The presence of non-permeant solutes, such as citrate, in the solution used for storage, prevents haemolysis of the phototreated, cation-permeable cells by counterbalancing the osmotic activity of haemoglobin.

Additive effects of dipyridamole and Trolox in protecting human red cells during photodynamic treatment.

BACKGROUND AND OBJECTIVES: Photodynamic treatment (PDT) of red blood cell (RBC) suspensions has been reported to result in virus inactivation, but also in deterioration of cell quality. Recently, we have demonstrated the potential usefulness of the reactive oxygen species scavenger dipyridamole in selectively protecting RBCs against the harmful side-effects of PDT. Unfortunately, dipyridamole-conferred protection against long-term photohaemolysis was incomplete. In the present study, dipyridamole was applied in combination with Trolox (a hydrophilic vitamin E analogue) in order to augment RBC protection. MATERIALS AND METHODS: Leucodepleted RBC suspensions (30% haematocrit) were treated with 1,9-dimethylmethylene blue (DMMB) and red light, and the effect of inclusion of dipyridamole and Trolox was assessed on potassium leakage as well as on short-term and long-term photohaemolysis. Possible interference of the scavenger cocktail with virus inactivation was examined using extracellular pseudorabies virus (PRV). RESULTS: Treatment of RBC with DMMB and red light resulted in enhanced potassium leakage and both short- and long-term haemolysis. Dipyridamole and Trolox showed additive protective effects against induction of potassium leakage and photohaemolysis, suggesting different protection mechanisms for the two scavengers. Combined inclusion of dipyridamole and Trolox did not interfere with efficacy of PRV inactivation. CONCLUSIONS: Combined inclusion of dipyridamole and Trolox results in substantially improved selectivity of photodynamic treatment of RBC suspensions.

Diversion of first blood volume results in a reduction of bacterial contamination for whole-blood collections.

BACKGROUND AND OBJECTIVES: In a previous study we established a reliable setpoint for the prevalence of bacteria in whole blood. In the present study we investigated the possible preventive effect, of diversion of the first 10 ml of a blood donation, on the bacterial contamination rate. MATERIALS AND METHODS: To divert the first 10 ml of a whole-blood donation, we used a special five-bag system equipped with a Composampler device. After venepuncture, the first 10 ml of a donation was sampled into a vacutainer tube. This was followed by the collection of the whole-blood unit. The extra bag allowed direct sampling of the final donation in a closed system for BacT/Alert. Whole-blood samples were taken after storage (2-14 h at 20 degrees C) and subsequent mixing. BacT/Alert culture bottles were incubated until positive, or for 7 days if negative. Confirmation and identification of positive cultures was performed according to internationally recognized standard reference methods. RESULTS: The prevalence of bacteria in whole blood, as determined by using standard collection techniques, was 0.35% (95% confidence interval 0.27-0.44%, n = 18 257). After diversion of the first 10 ml this value was significantly lower: 0.21% (P < 0.05, 95% confidence interval 0.12-0.35%, n = 7087). Most strikingly, a reduction in the frequency of staphylococcal species was observed (P < 0.02, reduction from 0.14 to 0.03%). CONCLUSIONS: Diversion of the first 10 ml of blood was shown to contribute significantly to a reduction in the prevalence of superficial skin bacteria in whole-blood units. In our opinion, blood collection systems should be adapted to use the first 10-30 ml of a whole-blood donation for testing purposes.

The band III ligand dipyridamole protects human RBCs during photodynamic treatment while extracellular virus inactivation is not affected.

BACKGROUND: Recently, the potential usefulness of dipyridamole (DIP) in protecting RBCs against the harmful side effects of photodynamic sterilization was demonstrated. In the present study, the use of DIP for selective protection of RBCs was investigated under conditions more relevant for blood bank practice. STUDY DESIGN AND METHODS: WBC-reduced RBC suspensions (30% Hct) were treated with 1,9-dimethylmethylene blue and red light, and the influence of the inclusion of DIP on photohemolysis was assessed as a function of sensitizer concentration, light dose, and storage time. Furthermore, the possible interference of DIP with inactivation of extracellular virus by use of a panel of different viruses (HIV-1, pseudorabies virus [PRV], bovine viral diarrhea virus [BVDV], VSV, encephalomyocarditis, and canine parvovirus) was investigated. RESULTS: In WBC-reduced RBC suspensions (30% Hct), DIP exerted a clear protective effect against photohemolysis. Part of this protection was achieved with concentrations near the dissociation constant for band III binding. Importantly, efficiency of inactivation of extracellular HIV-1, PRV, BVDV, and VSV was not significantly impaired by the inclusion of DIP. Phototreatment conditions, resulting in a 4 to 5 log inactivation of extracellular HIV-1 and PRV, resulted in a high level of hemolysis after 28 days of storage. This long-term hemolysis could be decreased, but not completely prevented, by the inclusion of DIP. CONCLUSION: Photohemolysis in RBC concentrates can be reduced substantially by the application of DIP, while the efficacy of inactivation of HIV-1 and other viruses remains unchanged.