Comparison of in vitro responses to fresh whole blood and reconstituted whole blood after collagen stimulation

Background

In patients who have large bleeds, there is a tendency to transfuse more plasma and platelets than recommended in earlier guidelines, and accordingly many hospitals now provide “transfusion packages” with an intended red cell:platelet:plasma ratio of 1:1:1. The purpose of this study was to investigate in vitro functions of transfusion packs compared with fresh whole blood.

Material and methods

“Reconstituted whole blood” was prepared with the same ratio of red cells, platelets and plasma as used in local transfusion packages. The aggregation and thrombin-antithrombin complex formation responses to collagen stimulation of this reconstituted whole blood were compared with those of fresh whole blood. The storage time of red cells and platelets was varied in a systematic manner, giving nine different compositions of reconstituted whole blood that simulated transfusion packs.

Results

The responses varied significantly between whole blood and reconstituted whole blood -and between the reconstituted whole blood of different compositions. A significant decrease (p<0.005) in collagen-induced platelet count reduction was seen with increasing platelet and red blood cell age. Thrombin-antithrombin complex formation peaked in studies with platelets stored for 5 days. The red cells stored for the longest time induced the greatest thrombin-antithrombin complex formation. Fresh whole blood gives more consistent responses, and the aggregation response to collagen is stronger than in reconstituted whole blood.

Discussion

Our results indicate that in vitro responses of reconstituted whole blood vary substantially according to how long the red cells and platelets are stored for. As the responses obtained by testing whole blood are more consistent and usually stronger, the alternative use fresh whole blood in special conditions should not be excluded without further consideration.     

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.     

Hypercoagulability in splenectomized thalassemic patients detected by whole-blood thromboelastometry, but not by thrombin generation in platelet-poor plasma

Background

The mechanisms responsible for the increased thrombotic risk associated with thalassemia are still unclear. They might be related to the effects of red blood cell or endothelial cell derangements, increased numbers of platelets as well as abnormal plasma coagulation.

Design and Methods

To evaluate the relative role played by cells and plasma we investigated 169 patients with thalassemia by means of thromboelastometry and thrombin generation tests. Thromboelastometry measures indices of the viscoelastic properties of whole blood after activation of coagulation and is characterized by the clotting time, which may be considered as a conventional coagulation time, clot formation time, defined as the time needed for the clot to reach a fixed firmness, and the maximum clot firmness, defined as the maximal amplitude of the tracing.

Results

All the thromboelastometry parameters determined in whole blood (including shortened clotting time and clot formation time, and increased maximum clot firmness), were consistent with hypercoagulability, especially in splenectomized patients. Conversely, thrombin generation as determined in platelet-poor plasma was not.

Conclusions

These findings point to blood cells and/or platelets rather than to plasma abnormalities as the most important determinants of the thrombotic risk observed in thalassemic patients who had been splenectomized. These results might have important diagnostic and therapeutic implications.     

A calcium-containing electrolyte-balanced hydroxyethyl starch (HES) solution is associated with higher factor VIII activity than is a non-balanced HES solution,but does not affect von Willebrand factor function or thromboelastometric measurements - results of a model of in vitro haemodilution

Background

Hydroxyethyl starch (HES) is known to impair blood coagulation. The impact of calcium-containing, balanced carrier solutions of HES on coagulation is controversial. We investigated the effects of increasing degrees of haemodilution with modern 6%, electrolyte-balanced HES vs non-balanced HES on coagulation in vitro, and compared the balanced HES to a balanced crystalloid solution for an internal control.

Materials and methods

Blood samples from ten healthy volunteers were diluted in vitro by 20%, 40% and 60% with either calcium-containing balanced 130/0.42 HES, non-balanced 130/0.4 HES or balanced crystalloid. In all samples, blood counts, prothrombin time ratio, activated partial thromboplastin time, ionized calcium, factor VIII activity, von Willebrand factor antigen, von Willebrand factor collagen binding activity, and von Willebrand factor activity were determined, and activated rotational thromboelastometry (EXTEM and FIBTEM assays) was performed.

Results

Haemodilution impaired coagulation in a dilution-dependent manner as determined by both conventional laboratory assays and thromboelastometry. Ionized calcium increased with balanced HES (p≤0.004), but decreased with non-balanced HES (p≤0.004). Prothrombin time ratio (p≤0.002) and factor VIII levels (p=0.001) were better preserved with balanced HES than with non-balanced HES in dilutions ≥40%. Thromboelastometry showed no differences between values in blood diluted with the balanced or non-balanced HES.

Discussion

In vitro, a balanced calcium-containing carrier solution of 6% HES 130/0.42 preserved coagulation better than did non-balanced HES 130/0.4 as quantified by conventional coagulation assays, but not in activated thromboelastometry. One explanation could be the increased ionized calcium levels after dilution with calcium-containing carrier solutions.     

The effects of pneumatic tube transport on fresh and stored platelets in additive solution

Background

Limited scientific work has been conducted on potential in vitro effects of transport on pneumatic tube systems on blood components, in particular platelets.

Materials and methods

To evaluate the possible effects of the Swisslog TranspoNet system on the cellular, metabolic, phenotypic and secreting properties of fresh and stored platelets, we set up a four-arm paired study comparing transported and non-transported platelets. Platelets were aliquoted, prepared with the OrbiSac system and suspended in 70% SSP+ (n=8). All in vitro parameters were monitored over a 7-day storage period.

Results

Throughout storage, no differences were observed in glucose consumption, lactate production, pH, pCO₂, ATP, hypotonic shock response reactivity, CD62P, PAC-1, platelet endothelial cell adhesion molecule-1 or CD42b. The release of sCD40L increased (p<0.01) in all units but without any significant differences between groups.

Conclusion

The storage stability of all platelets conveyed by the Swisslog TranspoNet system was not impaired throughout 7 days of storage. The Swisslog TranspoNet system does not, therefore, seem to be a risk for increased metabolic activity, activation or release reactions from the platelets. This lack of effect of the pneumatic tube transport system did not seem to be affected by the age of the platelets or repeated transport.     

Physiologically aged red blood cells undergo erythrophagocytosis in vivo but not in vitro

Background

The lifespan of red blood cells is terminated when macrophages remove senescent red blood cells by erythrophagocytosis. This puts macrophages at the center of systemic iron recycling in addition to their functions in tissue remodeling and innate immunity. Thus far, erythrophagocytosis has been studied by evaluating phagocytosis of erythrocytes that were damaged to mimic senescence. These studies have demonstrated that acquisition of some specific individual senescence markers can trigger erythrophagocytosis by macrophages, but we hypothesized that the mechanism of erythrophagocytosis of such damaged erythrocytes might differ from erythrophagocytosis of physiologically aged erythrocytes.

Design and Methods

To test this hypothesis we generated an erythrocyte population highly enriched in senescent erythrocytes by a hypertransfusion procedure in mice. Various erythrocyte-aging signals were analyzed and erythrophagocytosis was evaluated in vivo and in vitro.

Results

The large cohort of senescent erythrocytes from hypertransfused mice carried numerous aging signals identical to those of senescent erythrocytes from control mice. Phagocytosis of fluorescently-labeled erythrocytes from hypertransfused mice injected into untreated mice was much higher than phagocytosis of labeled erythrocytes from control mice. However, neither erythrocytes from hypertransfused mice, nor those from control mice were phagocytosed in vitro by primary macrophage cultures, even though these cultures were able to phagocytose oxidatively damaged erythrocytes.

Conclusions

The large senescent erythrocyte population found in hypertransfused mice mimics physiologically aged erythrocytes. For effective erythrophagocytosis of these senescent erythrocytes, macrophages depend on some features of the intact phagocytosing tissue for support.     

Measurement of Glucose in Blood with a Phenylboronic Acid Optical Sensor

Background

Current methods of glucose monitoring rely predominantly on enzymes such as glucose oxidase for detection. Phenylboronic acid receptors have been proposed as alternative glucose binders. A unique property of these molecules is their ability to bind glucose in a fully reversible covalent manner that facilitates direct continuous measurements. We examined (1) the ability of a phenylboronic-based sensor to measure glucose in blood and blood plasma and (2) the effect on measurement accuracy of a range of potential interferents. We also showed that the sensor is able to track glucose fluctuations occurring at rates mimicking those experienced in vivo.

Method

In vitro static measurements of glucose in blood and blood plasma were conducted using holographic sensors containing acrylamide, N,N′-methylenebisacrylamide, 3-acrylamidophenylboronic acid, and (3-acrylamidopropyl) trimethylammonium chloride. The same sensors were also used for in vitro measurements performed under flow conditions.

Results

The opacity of the liquid had no affect on the ability of the optical sensor to measure glucose in blood or blood plasma. The presence of common antibiotics, diabetic drugs, pain killers, and endogenous substances did not affect the measurement accuracy, as shown by error grid analysis. Ex vivo flow experiments showed that the sensor is able to track changes accurately in concentration occurring in real time without lag or evidence of hysteresis.

Conclusions

The ability of phenylboronic acid sensors to measure glucose in whole blood was demonstrated for the first time. Holographic sensors are ideally suited to continuous blood glucose measurements, being physically and chemically robust and potentially calibration free.     

Metabolic biofouling of glucose sensors in vivo: role of tissue microhemorrhages

Objective:

Based on our in vitro study that demonstrated the adverse effects of blood clots on glucose sensor function, we hypothesized that in vivo local tissue hemorrhages, induced as a consequence of sensor implantation or sensor movement post-implantation, are responsible for unreliable readings or an unexplained loss of functionality shortly after implantation.

Research Design and Methods:

To investigate this issue, we utilized real-time continuous monitoring of blood glucose levels in a mouse model. Direct injection of blood at the tissue site of sensor implantation was utilized to mimic sensor-induced local tissue hemorrhages.

Results:

It was found that blood injections, proximal to the sensor, consistently caused lowered sensor glucose readings, designated temporary signal reduction, in vivo in our mouse model, while injections of plasma or saline did not have this effect.

Conclusion:

These results support our hypothesis that tissue hemorrhage and resulting blood clots near the sensor can result in lowered local blood glucose concentrations due to metabolism of glucose by the clot. The lowered local blood glucose concentration led to low glucose readings from the still functioning sensor that did not reflect the systemic glucose level.     

In vitro evaluation of clot quality and stability in a model of severe thrombocytopenia: effect of fibrinogen,factor XIII and thrombin-activatable fibrinolysis inhibitor

Background

The treatment options in severe thrombocytopenia (platelet count ≤20×10⁹/L) are limited. The aim of this study was to investigate ways of improving blood clotting and stability in reconstituted thrombocytopenia.

Materials and methods

Thrombocytopenia (platelets [16±4]×10⁹/L) was created by differential centrifugation of normal blood followed by reconstitution of whole blood which was subjected to clotting in a rotation thromboelastometer by CaCl₂ and tissue factor, and to fibrinolysis by tissue plasminogen activator (tPA). In separate experiments, blood was diluted by 40% with TRIS/saline solution. Blood was treated with fibrinogen (fib), factor XIII (FXIII), and thrombin-activatable fibrinolysis inhibitor (TAFI).

Results

The maximum clot firmness of thrombocytopenic blood was approximately 2-fold less than that of intact blood. Supplementation of blood with fib and FXIII improved clot formation. In the presence of tPA, among fib, FXIII and TAFI, only fib stimulated clot propagation whereas each of these agents increased clot strength. There was a synergistic effect when fib was added together with FXIII or TAFI. Fibrinolysis was inhibited by TAFI and to a greater extent by TAFI + FXIII. Fourty percent dilution of blood reduced clot strength and increased susceptibility to tPA. Clot strength was increased by the treatments in the following order: fib/FXIII/TAFI > fib/TAFI > fib > TAFI > FXIII. In the presence of tPA, TAFI and FXIII lysed the clots significantly more slowly. This effect was stronger when blood was treated with the combination of fib/FXIII/TAFI. Doubling the fib concentration, alone or together with other agents, did not improve clot strength or stability.

Discussion

Augmentation of clot formation and anti-fibrinolysis by combining fib, FXIII and TAFI may be beneficial for the treatment of patients with severe thrombocytopenia especially when complicated by haemodilution following introduction of fluids to compensate for massive blood loss.     

Effect of haematocrit on fibrin-based clot firmness in the FIBTEM test

Background

Point-of-care thromboelastometry (ROTEM^®) can be used to assess coagulation in whole blood. In the ROTEM^® FIBTEM test, cytochalasin D eliminates the contribution of platelets to the whole blood clot; hence, only the remaining elements, including fibrinogen/fibrin, red blood cells and factor XIII, contribute to clot strength. We investigated the relationships between FIBTEM maximum clot firmness (MCF), whole blood fibrinogen concentration and plasma fibrinogen concentration to determine the impact of haematocrit on these parameters during cardiac surgery.

Materials and methods

The relationships between FIBTEM MCF and both whole blood fibrinogen concentration and plasma fibrinogen concentration (Clauss assay) were evaluated pre-operatively and after cardiopulmonary bypass/protamine administration in haematocrit-based subgroups.

Results

The study included 157 patients. The correlation coefficient rho between FIBTEM MCF and plasma fibrinogen concentration was 0.68 at baseline and 0.70 after protamine, while that between FIBTEM MCF and whole blood fibrinogen concentration was 0.74 at baseline and 0.72 after protamine (all P <0.001). In subgroup analyses based on haematocrit levels, pre-operative FIBTEM MCF and whole blood fibrinogen concentration were both significantly higher (P <0.05) for the lowest haematocrit subgroup, but plasma fibrinogen concentration was similar in all groups. After protamine, no significant differences were observed between the lowest haematocrit group and the other groups for any of the three parameters.

Conclusions

The effect of haematocrit on blood clotting is not reflected by plasma fibrinogen concentration, in contrast to FIBTEM MCF which incorporates the contribution of haematocrit to whole blood clot firmness. This effect does, however, appear to be negligible in haemodiluted patients.     

Automated processing of whole blood units: operational value and in vitro quality of final blood components

Background

The Community Transfusion Centre in Madrid currently processes whole blood using a conventional procedure (Compomat, Fresenius) followed by automated processing of buffy coats with the OrbiSac system (CaridianBCT). The Atreus 3C system (CaridianBCT) automates the production of red blood cells, plasma and an interim platelet unit from a whole blood unit. Interim platelet unit are pooled to produce a transfusable platelet unit. In this study the Atreus 3C system was evaluated and compared to the routine method with regards to product quality and operational value.

Materials and methods

Over a 5-week period 810 whole blood units were processed using the Atreus 3C system. The attributes of the automated process were compared to those of the routine method by assessing productivity, space, equipment and staffing requirements. The data obtained were evaluated in order to estimate the impact of implementing the Atreus 3C system in the routine setting of the blood centre. Yield and in vitro quality of the final blood components processed with the two systems were evaluated and compared.

Results

The Atreus 3C system enabled higher throughput while requiring less space and employee time by decreasing the amount of equipment and processing time per unit of whole blood processed. Whole blood units processed on the Atreus 3C system gave a higher platelet yield, a similar amount of red blood cells and a smaller volume of plasma.

Discussion

These results support the conclusion that the Atreus 3C system produces blood components meeting quality requirements while providing a high operational efficiency. Implementation of the Atreus 3C system could result in a large organisational improvement.     

Single-donor platelet apheresis: observational comparison of the new Haemonetics Universal Platelet protocol with the previous Concentrated Single Donor Platelet protocol

Background

The Haemonetics MCS^®+ cell separator is a device dedicated to the collection of leucoreduced single-donor platelets. The new Universal Platelet protocol has been introduced to improve the efficiency of apheresis and increase flexibility in the collection of leucoreduced platelets in combination with red blood cells and plasma. In this study we compared its performance with that of the previous Concentrated Single Donor Platelet protocol.

Materials and methods

This observational study had a within-subject design and involved 135 donors who underwent plateletapheresis with both protocols. The primary end-point was collection efficiency; secondary end-points were other performance indices, such as procedure time and collection rate. A satisfaction questionnaire was also administered to the 135 donors to evaluate opinions on duration, comfort and side-effects of donations with the two protocols. For each parameter of interest, we tested the difference between the two protocols within donors, using a one-sample t-test or exact McNemar’s test as appropriate.

Results

The collection efficiency of the Universal Platelet protocol was significantly higher than that of the Concentrated Single Donor Platelet protocol (58% vs 47%; p<0.0001). The Universal Platelet Protocol collected more platelets in less time, leading to a higher collection rate (6.5 vs 5.0×10⁹/min; p<0.0001). In general, donors found apheresis with the Universal Platelet protocol of equal duration or faster, of similar or greater comfort and with an equal number or fewer side effects, compared with the Concentrated Single Donor Platelet protocol.

Discussion

Our study endorses the use of the new Universal Platelet protocol in daily transfusion practice since it substantially improves collection efficiency in leucoreduced platelet procedures compared with the Concentrated Single Donor Platelet protocol. This technical improvement seems to be accompanied by equal or greater comfort for the donor.     

Detection of apoptosis-associated microRNA in human apheresis platelets during storage by quantitative real-time polymerase chain reaction analysis

Background

Platelet transfusion is an essential part of the treatment of a variety of conditions such as thrombocytopenia and qualitative platelet disorders. As indicated in previous reports, during in vitro storage, platelets undergo morphological and physiological changes collectively known as the platelet storage lesion. Apoptosis is a programmed process of cell death, which has been considered as an important cause of platelet storage lesion under the common storage conditions in standard blood banks. Platelets are anucleate blood cells, but contain significant amounts of microRNA (miRNA, miR), which may play an important role in the regulation of gene expression. Drawing on previously published reports on cell apoptosis, we selected 49 miRNA for analysis to explore whether miRNA are of importance during the storage of platelets.

Materials and methods

We used quantitative real-time polymerase chain reaction analysis to determine the levels of expression of miRNA in apheresis platelets at different times of storage. Bioinformatics analysis was applied to explore target genes and the main functions of the selected miRNA.

Results

Our observations suggest that apheresis platelets contain large amounts of apoptosis-associated miRNA. The levels of expression of 25 miRNA remained high and ten of these miRNA showed different expression from that at day 0. Of these ten miRNA, hsa-miR-326, hsa-miR-96, hsa-miR-16, hsa-miR-155 and hsa-miR-150 were up-regulated, while hsa-miR-7, hsa-miR-145, hsa-miR-24, hsa-miR-25 and hsa-miR-15a were down-regulated. The markedly increased expression of hsa-miR-326 in all platelets is noteworthy (p<0.001).

Discussion

Since Bcl-xl and Bak1, members of the Bcl-2 family, are the targets of hsa-miR-326, our findings suggest that hsa-miR-326 may be involved in platelet apoptosis during storage.     

Coagulopathy after a liver resection: is it over diagnosed and over treated?

Background

Prothrombin time-international normalized ratio (PT-INR) is widely utilized to guide plasma therapy and initiation of thromboprophylaxis after a hepatectomy. Thrombelastography (TEG) monitors shear elasticity, which is sensitive to cellular and plasma components in blood, allowing for functional assessment of the life of the clot. The objective of this study was to prospectively compare PT-INR and TEG in liver resection patients.

Methods

Forty patients were enrolled before undergoing an elective hepatectomy. Patients underwent a liver resection utilizing a low central venous pressure (CVP) anaesthetic technique and intermittent Pringle manoeuver. PT-INR and TEG were drawn prior to incision, post-operatively, and post-operative days 1, 3 and 5.

Results

All post-operative PT-INR values increased significantly when compared with pre-operative PT-INR (P < 0.01). The time of onset to clot (R-value) decreased significantly at the post-operative time point (P = 0.04), consistent with a relative hypercoagulability. Subsequent R-values were not different compared with the pre-operative R-value. The strength of the clot (maximum amplitude, MA) was unchanged when comparing pre- and post-operative time points.

Discussion

In spite of an elevation in PT-INR, patients undergoing a liver resection demonstrated a brief hypercoagulable state, followed by normal coagulation function based on TEG. These data call into question the practice of utilizing PT-INR to guide plasma transfusion and timing of prophylactic anticoagulation after a liver resection.     

Storage of whole blood overnight in different blood bags preceding preparation of blood components: in vitro effects on red blood cells

Background

Routines for the storage of whole blood (WB) overnight for the preparation of blood components on the following day are of increasing interest primarily for logistic reasons. The present study focuses on in vitro effects during storage for 6 weeks on red blood cells (RBC) prepared in different blood containers after being held overnight.

Study design and methods

Five different blood collection systems were used with either inline leucocyte reduction red cell filters for the preparation of RBC, buffy coat (BC) and plasma or WB filters for the preparation of RBC and plasma. A new container with an integrated WB filter removing leucocytes but not platelets was also included for the preparation of leucocyte-reduced RBC, BC and plasma units. Standard CPD solution (63 or 70 mL) and SAG-M solution (100 or 110 mL) were used for the collection of either 450 or 500 mL blood. All WB units were stored at room temperature, either overnight for 18–24 hours (test groups, n=104) or for up to 8 hours (reference groups, n=20). In addition, five test units were stored overnight under refrigeration.

Results

In test groups (overnight storage at room temperature) we found significantly lower levels of extracellular potassium, 2,3-DPG and pH (up to day 28). During storage, higher levels of ATP (Terumo, CaridianBCT until day 35, Fresenius until day 14, Fenwal throughout storage) were seen in test groups than in reference groups. When WB was stored overnight at 2–6°C before WB filtration, the levels of ATP and haemolysis were higher than in the corresponding reference.

Conclusion

Significant differences in in vitro parameters were observed between RBC prepared within 8 hours and 18–24 hours after blood collection. The results were consistent irrespective of the blood container used. New alkaline solutions may decrease the differences.     

Correction of subclinical coagulation disorders before percutaneous dilatational tracheotomy

Background

There is evidence that percutaneous dilatational tracheotomy (PDT) can be safely performed in patients with severe coagulation disorders if these are carefully corrected immediately before the procedure. However, it is currently unclear whether PDT can be performed safely in patients in an Intensive Care Unit (ICU) with uncorrected mild coagulation disorders.

Materials and methods

In a randomised controlled trial we determined the effect of correction of mild coagulation disorders on bleeding during and after PDT. ICU patients planned for bedside PDT with: (i) a prothrombin time (PT) between 14.7–20.0 seconds, (ii) a platelet count between 40–100×10⁹/L and/or (iii) active treatment with acetylsalicylic acid were randomised to receive infusion with fresh-frozen plasma (FFP) and/or platelets (“correction”) versus no transfusion (“no correction”) before PDT.

Results

We randomised 35 patients to the “correction” group and 37 patients to the “no correction” group. In patients who received FFP, the decrease in PT was marginal (mean decrease 0.40±0.56 seconds); the median increase in platelet counts after transfusion of platelets was 35 [11–47]x10⁹/L. The median blood loss was 3 [IQR: 1–6] grams in the “correction” group and 3 [IQR: 2–6] grams in the “no correction” group (P=0.96).

Discussion

Bleeding during and after bedside PDT in ICU patients with mild coagulation disorders is rare in our setting. Correction of subclinical coagulation disorders by transfusion of FFP and/or platelets does not affect bleeding.     

Heparanase enhances the generation of activated factor X in the presence of tissue factor and activated factor VII

Background

Heparanase is an endo-β-D-glucuronidase dominantly involved in tumor metastasis and angiogenesis. Recently, we demonstrated that heparanase is involved in the regulation of the hemostatic system. Our hypothesis was that heparanase is directly involved in activation of the coagulation cascade.

Design and Methods

Activated factor X and thrombin were studied using chromogenic assays, immunoblotting and thromboelastography. Heparanase levels were measured by enzyme-linked immunosorbent assay. A potential direct interaction between tissue factor and heparanase was studied by co-immunoprecipitation and far-western assays.

Results

Interestingly, addition of heparanase to tissue factor and activated factor VII resulted in a 3- to 4-fold increase in activation of the coagulation cascade as shown by increased activated factor X and thrombin production. Culture medium of human embryonic kidney 293 cells over-expressing heparanase and its derivatives increased activated factor X levels in a non-enzymatic manner. When heparanase was added to pooled normal plasma, a 7- to 8-fold increase in activated factor X level was observed. Subsequently, we searched for clinical data supporting this newly identified role of heparanase. Plasma samples from 35 patients with acute leukemia at presentation and 20 healthy donors were studied for heparanase and activated factor X levels. A strong positive correlation was found between plasma heparanase and activated factor X levels (r=0.735, P=0.001). Unfractionated heparin and an inhibitor of activated factor X abolished the effect of heparanase, while tissue factor pathway inhibitor and tissue factor pathway inhibitor-2 only attenuated the procoagulant effect. Using co-immunoprecipitation and far-western analyses it was shown that heparanase interacts directly with tissue factor.

Conclusions

Overall, our results support the notion that heparanase is a potential modulator of blood hemostasis, and suggest a novel mechanism by which heparanase increases the generation of activated factor X in the presence of tissue factor and activated factor VII.     

Use of blood products and risk of stroke after coronary artery bypass surgery

Background

The impact of blood transfusion on the development of post-operative stroke after coronary artery bypass grafting (CABG) is not well established. We, therefore, investigated this issue.

Materials and methods.

Complete data on peri-operative blood transfusion were available for 2,226 patients who underwent CABG in three Finnish hospitals.

Results

Stroke occurred post-operatively in 53 patients (2.4%). Logistic regression showed that pre-operative creatinine (OR 1.003, 95% CI 1.000–1.006), extracardiac arteriopathy (OR 2.344, 95% CI 1.133–4.847), pre-operative atrial fibrillation (OR 2.409, 95% CI 1.149–5.052), and the number of packed red blood cell units transfused (OR 1.121, 95% CI 1.065–1.180) were significantly associated with post-operative stroke. When the various blood product transfusions instead of transfused units were included in the multivariable analysis, solvent/detergent treated plasma (Octaplas^®) transfusion (OR 2.149, 95% CI 1.141–4.047), but not red blood cell transfusion, was significantly associated with postoperative stroke. Use of blood products ranging from no transfusion (stroke rate 1.6%) to combined transfusion of red blood cells, platelets and Octaplas^® was associated with a significant increase in post-operative stroke incidence (6.6%, adjusted analysis: OR 1.727, 95% 1.350–2.209). Patients who received >2 units of red blood cells, >4 units of Octaplas^® units and >8 units of platelets had the highest stroke rate of 21%. CART analysis showed that increasing amount of transfused Octaplas^®, platelets and history of extracardiac arteriopathy were significantly associated with post-operative stroke.

Conclusions

Transfusion of blood products after CABG has a strong, dose-dependent association with the risk of stroke. The use of Octaplas^® and platelet transfusions seem to have an even larger impact on the development of stroke than red blood cell transfusions.     

The effect of an instant hand sanitizer on blood glucose monitoring results

Background

People with diabetes mellitus are instructed to clean their skin prior to self-monitoring of blood glucose to remove any dirt or food residue that might affect the reading. Alcohol-based hand sanitizers have become popular when soap and water are not available. The aim of this study was to determine whether a hand sanitizer is compatible with glucose meter testing and effective for the removal of exogenous glucose.

Methods

We enrolled 34 nonfasting subjects [14 male/20 female, mean ages 45 (standard deviation, 9.4)] years, 2 with diagnosed diabetes/32 without known diabetes]. Laboratory personnel prepared four separate fingers on one hand of each subject by (1) cleaning the second finger with soap and water and towel drying (i.e., control finger), (2) cleaning the third finger with an alcohol-based hand sanitizer, (3) coating the fourth finger with cola and allowing it to air dry, and (4) coating the fifth finger with cola and then cleaning it with the instant hand sanitizer after the cola had dried. Finger sticks were performed on each prepared finger and blood glucose was measured. Several in vitro studies were also performed to investigate the effectiveness of the hand sanitizer for removal of exogenous glucose.z

Results

Mean blood glucose values from fingers cleaned with instant hand sanitizer did not differ significantly from the control finger (p = .07 and .08, respectively) and resulted in 100% accurate results. Blood glucose data from the fourth (cola-coated) finger were substantially higher on average compared with the other finger conditions, but glucose data from the fifth finger (cola-coated then cleaned with hand sanitizer) was similar to the control finger. The data from in vitro experiments showed that the hand sanitizer did not adversely affect glucose meter results, but when an exogenous glucose interference was present, the effectiveness of the hand sanitizer on glucose bias (range: 6% to 212%) depended on the surface area and degree of dilution.

Conclusions

In our study, use of an instant hand sanitizer was compatible with the results of a blood glucose monitor and did not affect finger stick blood glucose results. However, depending on surface area, hand sanitizers may not be adequate for cleaning the skin prior to glucose meter testing.     

Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model

Background

Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors.

Design and Methods

We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation.

Results

We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice.

Conclusions

These results demonstrate that human induced pluripotent stem cells: i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment.Key words: human induced pluripotent stem cells, terminal maturation, erythropoietic differentiation