Levels of factor VIII and factor IX in fresh-frozen plasma produced from whole blood stored at 4 °C overnight in Turkey

Background

The aim of this study was to assess whether the quantities of factor VIII and factor IX in fresh-frozen plasma produced from whole blood stored at 4 °C for 24 hours are adequate for their intended purpose.

Materials and methods

Fresh-frozen plasma separated from whole blood after storage at 4 °C overnight (24 hours from donation) was compared with plasma prepared 8 hours after donation using a standard method. The amounts of factor VIII and factor IX obtained with the two methods were compared.

Results

Compared to the levels of factor VIII and factor IX in plasma prepared within 8 hours of blood collection, the levels in plasma prepared after 24 hours of storage at 4 °C were 25% and 9% lower, respectively. Ninety percent of the factor VIII and 100% of the factor IX levels were above 0.5 IU/mL (standard haematology reference range) after 24 hours of storage.

Discussion

These data suggest that there is good retention of coagulation factor activity in plasma produced from whole blood stored at 4 ºC for 24 hours and that such plasma would be an acceptable product for most patients requiring fresh-frozen plasma.     

Effects of helicopter transport on red blood cell components

Background

There are no reported studies on whether a helicopter flight affects the quality and shelf-life of red blood cells stored in mannitol-adenine-phosphate.

Materials and methods

Seven days after donation, five aliquots of red blood cells from five donors were packed into an SS-BOX-110 container which can maintain the temperature inside the container between 2 °C and 6 °C with two frozen coolants. The temperature of an included dummy blood bag was monitored. After the box had been transported in a helicopter for 4 hours, the red blood cells were stored again and their quality evaluated at day 7 (just after the flight), 14, 21 and 42 after donation. Red blood cell quality was evaluated by measuring adenosine triphosphate, 2,3-diphosphoglycerate, and supernatant potassium, as well as haematocrit, intracellular pH, glucose, supernatant haemoglobin, and haemolysis rate at the various time points.

Results

During the experiment the recorded temperature remained between 2 and 6 °C. All data from the red blood cells that had undergone helicopter transportation were the same as those from a control group of red blood cell samples 7 (just after the flight), 14, 21, and 42 days after the donation. Only supernatant Hb and haemolysis rate 42 days after the donation were slightly increased in the helicopter-transported group of red blood cell samples. All other parameters at 42 days after donation were the same in the two groups of red blood cells.

Discussion

These results suggest that red blood cells stored in mannitol-adenine-phosphate are not significantly affected by helicopter transportation. The differences in haemolysis by the end of storage were small and probably not of clinical significance.     

Temporal sequence of major biochemical events during Blood Bank storage of packed red blood cells

Background.

We used sensitive spectroscopic techniques to measure changes in Band 3 oligomeric state during storage of packed red blood cells (RBC); these changes were compared to metabolic changes, RBC morphology, cholesterol and membrane protein loss, phospholipid reorganisation of the RBC membrane, and peroxidation of membrane lipid. The aim of the study was to temporally sequence major biochemical events occurring during cold storage, in order to determine which changes may underlie the structural defects in stored RBC.

Materials and methods.

Fifteen RBC units were collected from normal volunteers and stored under standard blood bank conditions; both metabolic changes and lipid parameters were measured by multiple novel assays including a new mass spectrometric measurement of isoprostane (lipid peroxidation) and flow cytometric assessment of CD47 expression. Band 3 oligomeric state was assessed by time-resolved phosphorescence anisotropy, and RBC morphology by microscopy of glutaraldehyde-fixed RBC.

Results.

Extracellular pH decreased and extracellular potassium increased rapidly during cold storage. Band 3 on the RBC membrane aggregated into large oligomers early in the storage period and coincident with changes in RBC morphology. Membrane lipid changes, including loss of unesterified cholesterol, lipid peroxidation and expression of CD47, also changed early during the storage period. In contrast loss of acetylcholinesterase activity and haemolysis of RBC occurred late during storage.

Discussion.

Our results demonstrate that changes in the macromolecular organisation of membrane proteins on the RBC occur early in storage and suggest that lipid peroxidation and/or oxidative damage to the membrane are responsible for irreversible morphological changes and loss of function during red cell storage.     

Effects of storage time and leucocyte burden of packed and buffy-coat depleted red blood cell units on red cell storage lesion

Background

The red cell storage lesion (RCSL) comprises the biochemical and biomechanical changes that take place during red blood cell (RBC) storage, reducing the survival and function of these cells. Contaminating white blood cells have been major contributors to the RCSL. Markers of RCSL, such as CD47 and phosphatidylserine (PS), on RBC are attracting more attention. The aim of this study was to elucidate the effects of storage time and buffy-coat removal on CD47 and PS expression on RBC. Potassium and free haemoglobin levels in the supernatant plasma were also assessed.

Materials and methods

Forty-three red cell concentrates were divided into two groups [Group 1: packed red cells (n=22); Group 2: red cell units from which the buffy-coat had been removed (n=21)] and samples were collected on days 1, 14 and 28. Flow cytometry was used to monitor changes of CD47 and PS expression on RBC over times. Supernatant potassium was measured and percent of haemolysis calculated.

Results

A significant, progressive decrease in RBC CD47 expression during storage was observed in both groups. The decrease in RBC CD47 expression was significantly less in the buffy-coat-removed group of units than in the other group. The percentage of annexin V-positive cells increased significantly in both groups. Buffy-coat depleted components showed less expression of PS only in the early samples. There were significant, progressive increases in percentage of haemolysis and supernatant potassium during storage in both groups.

Conclusion

RBC stored for more than 14 days exhibited reduced CD47 and increased PS. Buffy coat removal reduced the loss of CD47, but had no impact on plasma haemoglobin, potassium or RBC PS exposure.     

Coagulation factors V, VIII, and X, prothrombin time and activated partial thromboplastin time test results in thawed plasma stored at 1�C6 oC for 5 days

Background

The aim of this study was to evaluate whether thawed plasma stored for 5 days at 1–6 ºC is appropriate for use.

Materials and methods

Samples taken from thawed plasma units stored for 5 days at 1–6 ºC were prepared for assays of the levels of factor V, factor VIII, and factor X, and for measurements of the activated partial thromboplastin time (aPTT) and prothrombin time (PT). The results obtained were then compared with those from other similar studies.

Results

The average decreases in percentages of coagulation factors from day 1 to day 5 were 20% for factor V, 11% for factor X, and 25% for total factor VIII. The changes in the range of coagulation factors over the 5-day storage period were statistically significant, but the values remained within the haemostatic range. The average increases in the levels of PT and aPTT from day 1 to day 5 were also not clinically pathological.

Conclusions

Thawed fresh-frozen plasma stored for 5 days at 1–6 °C can be used in different cases of coagulopathy and can be used as an alternative to plasma frozen within 24 hours of phlebotomy.     

The age of red blood cells is associated with bacterial infections in critically ill trauma patients

Background.

Blood transfusion increases the risk of nosocomial infection in trauma patients. Specific patient- and transfusion-related risk factors are largely unknown. In this study, risk factors for developing a bacterial infection after transfusion of red blood cells (RBC) or platelets were determined in a cohort of transfused critically ill trauma patients.

Material and methods.

A retrospective study was conducted in a mixed medical-surgical Intensive Care Unit (ICU) of a level-1 university trauma centre, in trauma patients who received a RBC or platelet transfusion. Patients who developed a bacterial infection after transfusion were compared to transfused controls who did not develop such an infection. Multivariable logistic regression was used to determine risk factors for infection.

Results.

Of the 7,118 patients admitted to the ICU during the study period, 196 trauma patients met the inclusion criteria. An infection developed in 56 patients (29%). Infection occurred irrespective of the administration of antibiotics as part of selective digestive tract decontamination, surgery status or Injury Severity Score. Transfusion of RBC stored for more than 14 days was associated with infection in trauma patients (odds ratio 1.038, [95% CI: 1.01–1.07], p=0.036). Neither the amount of RBC nor that of platelets was associated with onset of infection.

Conclusions.

Transfusion of RBC stored for more then 14 days is a risk factor for onset of bacterial infection after trauma, irrespective of the use of prophylactic antibiotics. Transfusion of platelets was not a risk factor. These results may contribute to designing prospective studies on transfusion of fresh RBC only in trauma patients.     

Oxidative injury as contributory factor for red cells storage lesion during twenty eight days of storage

Background

Storage of red blood cells at 4 °C is associated with deleterious metabolic and biochemical changes, collectively referred to as “storage lesions”. Lipid peroxidation of the red cell membrane leading to lysis contributes to these storage lesions. The aim of the present study was to investigate oxidative injury to red cells during storage for 28 days and its correlation with markers of red cell membrane damage.

Materials and methods

Samples from 30 units of red blood cells stored at 4 °C for 28 days were withdrawn aseptically on day 0, day 14 and day 28 of storage. Markers of membrane damage including plasma haemoglobin, plasma potassium and lactate dehydrogenase (LDH) concentrations and markers of oxidative injury such as malondialdehyde (MDA) levels, haemoglobin oxidation and osmotic fragility were studied in all samples.

Results

Statistically significant (p<0.001) increases in the mean values of plasma haemoglobin, plasma potassium, LDH and markers of oxidative injury such as MDA and haemoglobin oxidation were observed over the storage period of 28 days. Direct correlations of MDA and haemoglobin oxidation with membrane damage, as reflected by plasma haemoglobin concentration, were observed.

Conclusion

Oxidative injury to red blood cells during storage leads to membrane damage and lysis. The role of antioxidants in the prevention of this deleterious effect of storage warrants investigation.     

Effects of cigarette smoke, nicotine and cotinine on red blood cell hemolysis and their -SH capacity

BACKGROUND:

Smoking is a leading cause of premature death. Red blood cell (RBC) membrane lipids are rich in polyunsaturated fatty acids; therefore, the effect of oxygen on RBC membranes is more prominent than on other body tissues. The attachment of peroxidants to RBC membranes can result in hemolysis.

OBJECTIVES:

The present study was conducted to assess the sensitivity of RBCs to 2,2′-azo-bis-(2-amidinopropane) dihydrochloride in smokers and nonsmokers. The effect of cigarette smoke, nicotine (1 μg/mL, 1.5 μg/mL and 2.5 μg/mL) and cotinine (1.25 μg/mL, 2.5 μg/mL and 5 μg/mL) on RBC hemolysis was also examined.

RESULTS:

RBC hemolysis in smokers was 21.6% higher than in non-smokers (P<0.05). Cigarette smoke increased 2,2′-azo-bis-(2-amidino-propane) dihydrochloride-induced RBC hemolysis by 281.7%. Nicotine inhibited RBC hemolysis by 36.7% at the highest concentration used, but increased RBC hemolysis at the lower concentrations. Cotinine caused a 13.8% increase in RBC membrane peroxidation at the highest concentration used and its effects were dose-dependent. At their highest concentrations, nicotine and cotinine decreased -SH groups by 50%.

CONCLUSIONS:

The present study confirms the results from previous studies of the oxidative and destructive effects of cigarette smoke, which are detrimental to the health of both active and passive smokers.     

Blood-Induced Interference of Glucose Sensor Function in Vitro: Implications for in Vivo Sensor Function

Background

Although tissue hemorrhages, with resulting blood clots, are associated with glucose sensor implantation, virtually nothing known is about the impact of red blood cells and red blood cell clots on sensor function in vitro or in vivo. In these studies, we tested the hypothesis that blood can directly interfere with glucose sensor function in vitro.

Methods

To test this hypothesis, heparinized human whole blood (HWB) and nonheparinized human whole blood (WB) were obtained from normal individuals. Aliquots of HWB and WB samples were also fractionated into plasma, serum, and total leukocyte (TL) components. Resulting HWB, WB, and WB components were incubated in vitro with an amperometric glucose sensor for 24 hours at 37°C. During incubation, blood glucose levels were determined periodically using a glucose monitor, and glucose sensor function (GSF) was monitored continuously as nanoampere output.

Results

Heparinized human whole blood had no significant effect on GSF in vitro, nor did TL, serum, or plasmaderived clots from WB. Sensors incubated with WB displayed a rapid signal loss associated with clot formation at 37°C. The half-life was 0.8 ± 0.2 hours (n = 16) for sensors incubated with WB compared to 3.2 ± 0.5 (n = 12) for sensors incubated with HWB with a blood glucose level of approximately 100 mg/dl.

Conclusions

These studies demonstrated that human whole blood interfered with GSF in vitro. These studies further demonstrated that this interference was related to blood clot formation, as HWB, serum, plasma-derived clots, or TL did not interfere with GSF in vitro in the same way that WB did. These in vitro studies supported the concept that the formation of blood clots at sites of glucose sensor implantation could have a negative impact on GSF in vivo.     

Peri-operative blood transfusion and operative time are quality indicators for pancreatoduodenectomy

Background

Minimization of blood loss during pancreatoduodenectomy requires careful surgical technique and specific preventative measures. Therefore, red blood cell (RBC) transfusions and operative time are potential surgical quality indicators. The aim of the present study was to compare peri-operative RBC transfusion and operative time with 30-day morbidity/mortality after pancreatoduodenectomy.

Methods

All pancreatoduodenectomies (2005 to 2008) were identified using the American College of Surgeons-National Surgical Quality Improvement Program (ACS-NSQIP). RBC transfusions and operative time were correlated with 30-day morbidity/mortality.

Results

Pancreatoduodenectomy was completed in 4817 patients. RBC transfusions were given to 1559 (32%) patients (1–35 units). Overall morbidity and mortality rates were 37% and 3.0%, respectively. Overall 30-day morbidity increased in a stepwise manner with the number of RBC transfusions (R =0.69, P < 0.01). Although RBC transfusions and operative times were not statistically linked (P =0.87), longer operative times were linearly associated with increased 30-day morbidity (R =0.79, P < 0.001) and mortality (R =0.65, P < 0.01). Patients who were not transfused also displayed less morbidity (33%) and mortality (1.9%) (P < 0.05).

Discussion

Peri-operative RBC transfusion after pancreatoduodenectomy is linearly associated with 30-day morbidity. Longer operative time also correlates with increased morbidity and mortality. Therefore, blood transfusions and prolonged operative time should be considered quality indicators for pancreatoduodenectomy.     

Overnight versus 24 Hours of Continuous Subcutaneous Insulin Infusion as Supplement to Oral Antidiabetic Drugs in Type 2 Diabetes

Background

Basal continuous subcutaneous insulin infusion (CSII) therapy at a fixed rate may effectively improve glycemic control in patients with type 2 diabetes when oral antidiabetic treatment fails. Regimens of simple constant subcutaneous delivery of insulin may provide theoretical advantages in type 2 diabetes.

Methods

Ten subjects with type 2 diabetes who obtained insufficient glycemic control on oral antidiabetic drugs were included. Following an initial control day, two periods of 3 days with CSII of a rapid-acting insulin analogue, 1.5 IU/h (dose obtained from a preceding study), for 8 hours overnight and for 24 hours, respectively, were carried out in random order. Profiles of serum insulin aspart, serum endogenous insulin, and plasma glucose were recorded.

Results

Compared to the control day, an 8-hour overnight insulin infusion during a 3-day period improved fasting plasma glucose (FPG) (mean differences ± SEM; Δ59.0 ± 10.1 mg/dl; p < 0.01) and 2-hour postprandial plasma glucose (PPPG) (Δ57.8 ± 10.6 mg/dl; p < 0.01) after breakfast. Compared to an 8-hour overnight infusion, a 24-hour infusion further improved all three PPPG values after breakfast, lunch, and dinner (Δ28.8 ± 8.1 mg/dl, Δ30.6 ± 8.1 mg/dl, and Δ35.1 ± 7.9 mg/dl; p < 0.01). During insulin infusion, only one hypoglycemic episode with PG <55.8 mg/dl and mild symptoms was recorded.

Conclusion

Continuous subcutaneous insulin infusion with a rapid-acting insulin analogue at a fixed rate of 1.5 IU/h, either overnight or for 24 hours, improved glycemic control without safety concerns in patients with type 2 diabetes who had secondary failure to oral antidiabetic drugs. The effect on FPG was similar for both treatments, whereas the effect on PPPG was superior when insulin was infused during the entire 24 hours.     

Biochemical assessment of red blood cells during storage by 1H nuclear magnetic resonance spectroscopy. Identification of a biomarker of their level of protection against oxidative stress

Background

Blood transfusion is an established therapeutic practice. The characteristics of blood components at different storage times are expected to affect the efficacy of transfusion therapy. Metabolic profiling by nuclear magnetic resonance (NMR) spectroscopy requires little or no sample treatment and allows identification of more than 50 soluble metabolites in a single experiment. The aim of this study was to assess the metabolic behaviour of red blood cells during 42 days of storage in blood bank conditions.

Materials and methods

Red blood cells (RBC), collected from eight healthy male donors, aged 25–50 years, were prepared as prestorage leukoreduced erythrocyte concentrates and stored under standard blood bank conditions. Samples taken at various storage times were separated in two fractions: the supernatant, recovered after centrifugation, and the red blood cell lysate obtained after protein depletion by ultrafiltration. The metabolic profile of the red blood cells was determined from analysis of ¹H-NMR spectra.

Results

The red blood cell supernatant was studied to track the consumption of the preservative additives and to detect and quantify up to 30 metabolites excreted by the erythrocytes. The NMR spectra of the RBC lysate provided complementary information on some biochemical pathways and set the basis for building a time-dependent red blood cell metabolic profile.

Discussion

We proved the analytical power of ¹H-NMR spectroscopy to study red blood cell metabolism under blood bank conditions. A potential biomarker able to provide information on the level of cellular oxidative stress protection was identified. Our data support the hypothesis that a more detailed knowledge of metabolic modifications during storage opens the way to the development of new and more effective protocols for red blood cell conservation and patient-oriented transfusion therapy.     

Validation and development of an immunonephelometric assay for the determination of alpha-1 antitrypsin levels in dried blood spots from patients with COPD

OBJECTIVE:

To validate and develop an immunonephelometric assay for the determination of alpha-1 antitrypsin (AAT) levels in dried blood spots from COPD patients in Brazil.

METHODS:

We determined AAT levels in serum samples and dried blood spots from 192 COPD patients. For the preparation of dried blood spots, a disk (diameter, 6 mm) was placed into a tube, eluted with 200 µL of PBS, and stored overnight at 4ºC. All of the samples were analyzed by immunonephelometry in duplicate. We used the bootstrap resampling method in order to determine a cut-off point for AAT levels in dried blood spots.

RESULTS:

The correlation coefficient between the AAT levels in serum samples and those in dried blood spots was r = 0.45. For dried blood spots, the cut-off value was 2.02 mg/dL (97% CI: 1.45-2.64 mg/dL), with a sensitivity, specificity, positive predictive value, and negative predictive value of 100%, 95.7%, 27.2%, and 100%, respectively.

CONCLUSIONS:

This method for the determination of AAT levels in dried blood spots appears to be a reliable screening tool for patients with AAT deficiency.     

Sample stability for complete blood cell count using the Sysmex XN haematological analyser

Background

Sample stability is a crucial aspect for the quality of results of a haematology laboratory. This study was conducted to investigate the reliability of haematological testing using Sysmex XN in samples stored for up to 24 h at different temperatures.

Materials and methods

Haematological tests were performed on whole blood samples collected from 16 ostensibly healthy outpatients immediately after collection and 3 h, 6 h or 24 h afterwards, with triple aliquots kept at room temperature, 4 °C or 37 °C.

Results

No meaningful bias was observed after 3 h under different storage conditions, except for red blood cell distribution width (RDW) and platelet count (impedance technique, PLT-I) at 37 °C. After 6 h, meaningful bias was observed for mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) at room temperature, red blood cell (RBC) count, mean corpuscular haemoglobin concentration (MCHC), MCH, MCV and PLT-I at 4 °C, and RBC, RDW, MCHC, MCH and PLT-I at 37 °C. After 24 h, a meaningful bias was observed for MCHC, MCV, platelet count (fluorescent technique, PLT-F) and mean platelet volume (MPV) at room temperature, MCHC, MCV, PLT-I and MPV at 4 °C, and all parameters except RBC count and MPV at 37 °C.

Discussion

Great caution should be observed when analysing results of haematological tests conducted more than 3 h after sample collection.     

Neonatal red blood cell transfusions: searching for better guidelines

Background

Packed red blood cell (RBC) transfusions are often administered to patients in the neonatal intensive care unit. The purpose of this study was to determine whether current blood transfusion guidelines are as useful as care givers’ perception in identifying patients in need of a packed RBC transfusion.

Design and Methods

Health care providers were asked to complete a pre- and post-transfusion survey on neonates receiving a packed RBC transfusion. These patients were divided into three groups based on reasons for transfusion: (i) guidelines; (ii) care-givers’ perceptions of need for packed RBC transfusion; or (iii) both. These three groups were further subdivided into two cohorts according to whether they had a clinical improvement or not. Demographic data and clinical variables were compared between the groups.

Results

Seventy-eight care-givers were surveyed. Eighteen patients (23%) were transfused based on guidelines, 36 (46%) based on care givers’ perception and 24 (31%) based on both. Neonates transfused based on guidelines alone were more likely to have received the transfusion in the first week of life, had a higher pre-transfusion haematocrit, were less symptomatic and had a higher trend to require mechanical ventilation. Neonates transfused based on caregivers’ perception were more likely to be on non-invasive ventilatory support and were more symptomatic. Neonates who improved after a transfusion had a lower pre-transfusion haematocrit (p=0.02), were more symptomatic (p=0.01) and were more likely to be on non-invasive ventilatory support (p=0.002) when compared to the group without a clinical improvement. The group without improvement had an increase in oxygen requirement (+2.8±6.4) after the transfusion (p=0.0004). Tachycardia was the most sensitive predictor of a benefit from packed RBC transfusion [OR 6.48: p=0.005].

Discussion

Guidelines on when to transfuse stable growing neonates with packed RBC should be re-evaluated to include more care giver judgement and perhaps be more restrictive for critically ill neonates.     

The influence of storage age on iron status,oxidative stress and antioxidant protection in paediatric packed cell units

Background

Receipt of blood transfusions is associated with the major consequences of prematurity such as bronchopulmonary dysplasia. Transfusion-mediated (iron-induced) oxidative damage, coupled with the limited ability of the premature baby to deal with enhanced iron and oxidative load may contribute to this. Adverse effects of transfusion may be related to duration of storage. This study examined the influence of storage on iron and oxidative status in paediatric packed red blood cell units.

Materials and methods

Paediatric packed red blood cell units were sampled 3 days post-donation, then at 7 days and weekly until day 35. The extracellular medium was separated and the following measured: total iron concentration, total iron binding capacity, non-transferrin-bound iron, haemoglobin, total and reduced ascorbate, glutathione and malondialdehyde.

Results

Measurable total and non-transferrin bound iron were present in the extracellular fluid of paediatric packs on day 3. Both parameters rose almost linearly to maximal values at 35 days. Haemoglobin and malondialdehyde levels rose gradually from day 3 to day 21, then more steeply to day 35. Ascorbate existed mainly in the oxidised form and fell rapidly towards the end of storage. Intracellular GSH fell throughout the period of storage. Strong correlations existed between biomarkers of oxidative damage and iron parameters.

Discussion

These data suggest that iron released following the initial preparation of packed red blood cell units may derive from free radical-mediated oxidative damage to the red blood cells and haemoglobin, rather than from extracellular haemoglobin. Iron continues to be released during storage as antioxidant protection declines. A cycle of free radical-mediated damage may initiate and then further exacerbate iron release during storage which, in turn, may mediate further free radical-mediated cellular damage. The potential consequences to recipients of older stored blood may be significant.     

Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

Background

Gene therapy provides an attractive alternative for protein replacement therapy in hemophilia A patients. Recent studies have shown the potential benefit of directing factor (F)VIII gene delivery to cells that also express its natural carrier protein von Willebrand factor (VWF). In this study, we explored the feasibility of blood outgrowth endothelial cells as a cellular FVIII delivery device with particular reference to long-term production levels, intracellular storage in Weibel-Palade bodies and agonist-induced regulated secretion.

Design and Methods

Human blood outgrowth endothelial cells were isolated from peripheral blood collected from healthy donors, transduced at passage 5 using a lentiviral vector encoding human B-domain deleted FVIII-GFP and characterized by flow cytometry and confocal microscopy.

Results

Blood outgrowth endothelial cells displayed typical endothelial morphology and expressed the endothelial-specific marker VWF. Following transduction with a lentivirus encoding FVIII-GFP, 80% of transduced blood outgrowth endothelial cells expressed FVIII-GFP. Levels of FVIII-GFP positive cells declined slowly upon prolonged culturing. Transduced blood outgrowth endothelial cells expressed 1.6±1.0 pmol/1×10⁶ cells/24h FVIII. Morphological analysis demonstrated that FVIII-GFP was stored in Weibel-Palade bodies together with VWF and P-selectin. FVIII levels were only slightly increased following agonist-induced stimulation, whereas a 6- to 8-fold increase of VWF levels was observed. Subcellular fractionation revealed that 15–22% of FVIII antigen was present within the dense fraction containing Weibel-Palade bodies.

Conclusions

We conclude that blood outgrowth endothelial cells, by virtue of their ability to store a significant portion of synthesized FVIII-GFP in Weibel-Palade bodies, provide an attractive cellular on-demand delivery device for gene therapy of hemophilia A.     

Evaluation of platelet function during extended storage in additive solution, prepared in a new container that allows manual buffy-coat platelet pooling and leucoreduction in the same system

Background

A novel and practical storage container designed for manual buffy-coat pooling and leucodepletion was evaluated to assess its filtration performance and to analyse the quality of stored leucoreduced buffy-coat-derived platelet pools.

Materials and methods.

To analyse the Grifols Leucored^® Transfer PL system, blood was collected from random donors into standard triple bag systems, and fractionated using standard procedures to obtain buffy-coats. Ten leucodepleted platelet pools were prepared each from five units of buffy-coats in additive solution. Concentrates were stored for 10 days at 22 °C on an end-over-end agitator. On days 0, 5, 7, and 10 of storage, samples were tested using standard in vitro platelet parameters.

Results

The use of this novel system for volume reduction and leucodepletion of buffy-coats resuspended in additive solution led to platelet pools that met the European requirements. pH was maintained well, declining from an initial value of 7.11±0.04 to 6.88±0.08 after 10 days. Parameters of cell lysis, response to a hypotonic stimulus and aggregation induced by agonists (arachidonic acid, ristocetin, collagen or thrombin receptor activating peptide) were also well-preserved. During storage, the quality profile of the platelet pools remained very similar to that previously reported in platelet concentrates in terms of metabolism, platelet activation (CD62, CD63, sCD62), expression of glycoproteins Ib and IIb/IIIa, capacity of glycoprotein IIb/IIIa to become activated upon ADP stimulation, and release of biological response modifiers (sCD40L and RANTES).

Discussion.

This new system allows the preparation of leucodepleted buffy-coat platelet pools in additive solution with good preservation of platelet function. The logistics of the procedure are relatively simple and it results in good-quality components, which may reduce costs and ease the process of buffy-coat pooling and leucocyte reduction in transfusion services.     

A comparative study of the effects of temperature, time and factor VIII assay type on factor VIII activity in cryoprecipitate in Iran

Background

In Iran, cryoprecipitate is an important plasma product to provide coagulation factors such as factor VIII (FVIII) in patients with factor VIII deficiency. FVIII is one of the labile coagulation factors and as such is also used as a quality marker of fresh-frozen plasma and cryoprecipitate. It is, therefore, important to optimise plasma production in order to prevent a reduction of FVIII activity. In this study we assessed the effect of temperature, time and FVIII assay type on FVIII activity in cryoprecipitate produced in Iran.

Methods

Ninety-six whole blood units were kept at two different temperatures (48 units kept at 1–6 °C and 48 kept at 20–24 °C) for periods of 4, 6, 8 or 10 hours before plasma freezing. FVIII activity was then measured by both chromogenic and one-stage clotting assays.

Results

At both temperatures, FVIII activity in plasma prepared after 8 and 10 hours was lower than that in plasma prepared after 4 and 6 hours. A significant decrease of FVIII activity was not seen in samples kept for 4 and 6 hours. Compared to storage between 1–6 °C, storage at 20–24 °C appears to cause a reduction in FVIII activity. There was a significant difference in apparent FVIII activity measured by the one-stage clot-based and chromogenic assays.

Conclusion

In Iran, to improve cryoprecipitate quality, freezing should begin within 6 hours after donation and whole blood should be kept at 1–6 °C until the plasma can be frozen. In this study although a good correlation was seen between the results of the one-stage clot-based and chromogenic assays for measuring FVIII activity in cryoprecipitate, the absolute values were significantly different.     

Low nocturnal blood pressure is associated with reduced cerebral blood flow in the cohort "Men born in 1914"

BACKGROUND:

“Men born in 1914” is a population-based cohort study of the epidemiology of cardiovascular and cerebral disease. Little is known about how diurnal variation in blood pressure (BP) levels influence cerebral perfusion in very elderly populations.

OBJECTIVES:

To study the association between systolic (SBP) and diastolic BP (DBP) levels, during the day and at night, expressed through 24 h ambulatory BP monitoring (ABPM) and regional cerebral blood flow (rCBF) disturbances.

METHODS:

A cross-sectional study from a population-based cohort of 108 men 81 years of age (born in 1914) was performed in an out-patient university clinic. Cerebral blood flow measurements using ^99mTc-hexamethylpropyleneamine oxime single photon emission computed tomography and 24 h ABPM were performed. Eleven men were excluded due to incomplete ABPM data.

RESULTS:

Mean DBP at night for each tertile was correlated to rCBF for the medial temporal right (P=0.012) and left (P=0.039) regions. Also, DBP during the day was correlated to the medial temporal right region (P=0.025). When analyses were stratified for DBP during the day, subjects with high DBP during the day (greater than 70 mmHg) showed a stronger association between low medial temporal right rCBF and low mean DBP at night (r=0.32, P=0.009) compared with subjects who had a lower daytime DBP. A corresponding positive correlation was noted for the medial temporal left region and daytime SBP, whereas a negative correlation was noted for frontal left region blood flow and SBP at night.

CONCLUSIONS:

A significant association was seen between low BP levels, especially at night, and rCBF in subjects with otherwise normal daytime DBP that may indicate a risk for nocturnal cerebral ischemia.