Repeated Blood Withdrawals Research Articles

Exploring the Relationship Between Serum Creatinine and Salivary Creatinine Levels in Patients With Chronic Kidney Disease in South India: A Cross-Sectional Study.

Background In this study, researchers investigated non-invasive methods for analyzing creatinine levels by using saliva to address the need for frequent phlebotomy in chronic kidney disease (CKD) patients, which can damage their veins due to repeated blood withdrawals for creatinine level assessments. Methods This is a cross-sectional study in a tertiary healthcare setting conducted on 50 patients diagnosed with CKD. After collecting serum and salivary creatinine, we used Pearson correlation to assess the correlation between the two factors. Results The mean age of the patients was 50 years with a standard deviation of ± 15.32 years. 33 (66%) patients were males and 17 (34%) were females. Most patients were in the age group of 51 - 70 years, comprising 26 (52%) of the sample. The serum creatinine and salivary creatinine values ranged between 7.26-12.00 and 0.45-0.98, respectively. The median values were 9.72 and 0.75, respectively. There was a very weak positive linear relationship between serum and salivary creatinine levels; however, there was no significant association between them (p = 0.52). Nonetheless, a statistically significant, moderately negative linear correlation exists between serum urea and serum albumin (r = -0.36; p = 0.01). Additionally, there is a statistically significant weak negative linear correlation between serum chloride and serum urea (r = -0.3; p = 0.03). Comparing serum chloride and serum sodium reveals a statistically significant, moderately positive linear relationship (r = 0.4; p = 0.004). Serum phosphorus and serum creatinine display a statistically significant moderate positive linear relationship (r = 0.44; p = 0.001). Moreover, estimated glomerular filtration rate (eGFR) and serum creatinine exhibit a statistically significant strong negative linear correlation (r = -0.79; p < 0.001), while eGFR and serum phosphorus demonstrate a statistically significant weak negative linear correlation (r = -0.30; p = 0.03). Conclusion The study found no significant association between salivary and serum creatinine levels. Further multicentric studies on a larger population must be conducted to find the potential correlation between serum and salivary markers.

Salivary Biomarkers of Anti-Epileptic Drugs: A Narrative Review.

Saliva is a biofluid that reflects general health and that can be collected in order to evaluate and determine various pathologies and treatments. Biomarker analysis through saliva sampling is an emerging method of accurately screening and diagnosing diseases. Anti-epileptic drugs (AEDs) are prescribed generally in seizure treatment. The dose-response relationship of AEDs is influenced by numerous factors and varies from patient to patient, hence the need for the careful supervision of drug intake. The therapeutic drug monitoring (TDM) of AEDs was traditionally performed through repeated blood withdrawals. Saliva sampling in order to determine and monitor AEDs is a novel, fast, low-cost and non-invasive approach. This narrative review focuses on the characteristics of various AEDs and the possibility of determining active plasma concentrations from saliva samples. Additionally, this study aims to highlight the significant correlations between AED blood, urine and oral fluid levels and the applicability of saliva TDM for AEDs. The study also focuses on emphasizing the applicability of saliva sampling for epileptic patients.

Chronic anemia is associated with systemic endothelial dysfunction.

In acute myocardial infarction and heart failure, anemia is associated with adverse clinical outcomes. Endothelial dysfunction (ED) is characterized by attenuated nitric oxide (NO)-mediated relaxation responses which is poorly studied in chronic anemia (CA). We hypothesized that CA is associated with ED due to increased oxidative stress in the endothelium. CA was induced by repeated blood withdrawal in male C57BL/6J mice. Flow-Mediated Dilation (FMD) responses were assessed in CA mice using ultrasound-guided femoral transient ischemia model. Tissue organ bath was used to assess vascular responsiveness of aortic rings from CA mice, and in aortic rings incubated with red blood cells (RBCs) from anemic patients. In the aortic rings from anemic mice, the role of arginases was assessed using either an arginase inhibitor (Nor-NOHA) or genetic ablation of arginase 1 in the endothelium. Inflammatory changes in plasma of CA mice were examined by ELISA. Expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS), myeloperoxidase (MPO), 3-Nitrotyrosine levels, and 4-Hydroxynonenal (4-HNE) were assessed either by Western blotting or immunohistochemistry. The role of reactive oxygen species (ROS) in ED was assessed in the anemic mice either supplemented with N-Acetyl cysteine (NAC) or by in vitro pharmacological inhibition of MPO. The FMD responses were diminished with a correlation to the duration of anemia. Aortic rings from CA mice showed reduced NO-dependent relaxation compared to non-anemic mice. RBCs from anemic patients attenuated NO-dependent relaxation responses in murine aortic rings compared to non-anemic controls. CA results in increased plasma VCAM-1, ICAM-1 levels, and an increased iNOS expression in aortic vascular smooth muscle cells. Arginases inhibition or arginase1 deletion did not improve ED in anemic mice. Increased expression of MPO and 4-HNE observed in endothelial cells of aortic sections from CA mice. NAC supplementation or inhibition of MPO improved relaxation responses in CA mice. Chronic anemia is associated with progressive endothelial dysfunction evidenced by activation of the endothelium mediated by systemic inflammation, increased iNOS activity, and ROS production in the arterial wall. ROS scavenger (NAC) supplementation or MPO inhibition are potential therapeutic options to reverse the devastating endothelial dysfunction in chronic anemia.

Combination therapy using fibrinogen γ-chain peptide-coated, ADP-encapsulated liposomes and hemoglobin vesicles for trauma-induced massive hemorrhage in thrombocytopenic rabbits.

We previously developed substitutes for red blood cells (RBCs) and platelets (PLTs) for transfusion. These substitutes included hemoglobin vesicles (HbVs) and fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV, H12)-coated, adenosine diphosphate (ADP)-encapsulated liposomes [H12-(ADP)-liposomes]. Here, we examined the efficacy of combination therapy using these substitutes instead of RBC and PLT transfusion in a rabbit model with trauma-induced massive hemorrhage with coagulopathy. Thrombocytopenia (PLT count approximately 40,000/μL) was induced in rabbits by repeated blood withdrawal and isovolemic transfusion with autologous RBCs. Thereafter, lethal hemorrhage was induced in rabbits by noncompressible penetrating liver injury. Subsequently, H12-(ADP)-liposomes with platelet-poor plasma (PPP), platelet-rich plasma (PRP), or PPP alone were administered to stop bleeding. Once achieving hemostasis, HbVs, allogenic RBCs, or 5% albumin were transfused into rabbits to rescue them from fatal anemia following massive hemorrhage. Administration of H12-(ADP)-liposomes/PPP as well as PRP (but not PPP) effectively stopped liver bleeding (100% hemostasis). The subsequent administration with HbVs as well as RBCs after hemostasis markedly rescued rabbits from fatal anemia (75% and 70% survivals for 24 hr, respectively). In contrast, 5% albumin administration rescued none of the rabbits. Combination therapy with H12-(ADP)-liposomes and HbVs may be effective for damage control resuscitation of trauma-induced massive hemorrhage.

Efficacy of Resuscitative Transfusion With Hemoglobin Vesicles in the Treatment of Massive Hemorrhage in Rabbits With Thrombocytopenic Coagulopathy and Its Effect on Hemostasis by Platelet Transfusion.

We have developed hemoglobin vesicles (HbVs) as a substitute for red blood cells (RBCs). We investigated the efficacy of HbV transfusion in the treatment of massive hemorrhage in rabbits in the setting of thrombocytopenic coagulopathy, focusing on the efficacy of hemostasis by subsequent platelet transfusion. Thrombocytopenic coagulopathy was induced in rabbits by repeated blood withdrawal and isovolemic retransfusion of autologous RBC (platelet counts <45,000/μL). A penetrating liver injury was then made. For 30 min, bleeding volume was measured every 10 min, after which subjects were transfused with an equivalent volume of stored RBC, HbV, or platelet poor plasma (PPP) to compensate for blood loss, simulating initial prehospital resuscitation. Thereafter, we transfused platelet rich plasma (PRP) to stop bleeding, which simulated inhospital resuscitation. During the initial resuscitation, the HbV group was similar to the RBC group (but not the PPP group) in their hemodynamics and tissue circulation/oxygenation as assessed by plasma lactate levels. All rabbits showed similar bleeding volumes (20-30 mL) in this period. HbV-transfused rabbits sustained hemoglobin levels, but showed lower hematocrit levels compared with RBC-transfused rabbits. Subsequent PRP transfusion effectively stopped bleeding in all RBC-transfused rabbits (6/6) and most HbV-transfused rabbits (7/8) but not PPP-transfused rabbits (2/8). In addition, 83% of RBC-transfused rabbits and 75% of HbV-transfused rabbits survived for 24 h, although no PPP-transfused rabbits survived. HbV transfusion did not scavenge nitric oxide in rabbits. HbV transfusion effectively rescued rabbits from severe hemorrhage with coagulopathy, without disturbing hemostasis after the platelet transfusion. HbV transfusion may be practical and useful in prehospital resuscitation.

The value of differential time to positivity of blood cultures in diagnosis of catheter-related bloodstream infection in patients with solid tumors in intensive care unit

To determine the value of differential time to positivity (DTTP) of blood culture for the diagnosis of catheter-related bloodstream infection (CRBSI) in patients with solid tumors in intensive care unit (ICU). A retrospective study was conducted. 615 pairs of peripheral vein blood cultures and instantaneous catheter tip blood culture of 615 patients admitted to ICU of Tianjin Medical University Cancer Institute and Hospital were collected from August 2011 to March 2014. The DTTP method and (or) semi quantitative culture of catheter tip were compared. CRBSI was diagnosed when both cultures were positive for the same microorganism and DTTP≥2 hours (120 minutes). The result of this procedure was compared with that of organism obtained using the semi quantitative culture of blood at catheter tip with ≥15 cfu. Based on the clinical diagnosis, the reliability of two kinds of laboratory examination was compared for the diagnosis of CRBSI by plotting receiver operator characteristic curve (ROC curve). The result of 615 cases suspected of having CRBSI were analyzed during the study period. Of these, 440 episodes were excluded because cultures were negative for blood obtained through peripheral vein and central vein. Eight episodes were excluded because only peripheral vein blood culture was positive and 57 episodes were excluded because of only central vein blood culture was positive, 68 pairs of blood cultures were excluded due to the presence of multiple catheters and repeated blood withdrawals. Two cases of polymicrobial cultures were excluded from the final analysis due to the difficulty in determining the time of positive result for each individual microorganism. Ten cases in 42 cases of suspected cases of CRBSI were excluded from analysis because catheter was not removed, therefore culture from catheter tip could not be obtained. Using the DTTP method, 14 out of 17 CRBSI cases were diagnosed with DTTP≥120 minutes, while 3 cases were missed; the semi quantitative catheter tip culture was positive in 13 cases, and in 4 cases it was neglected. In 2 cases of CRBSI it was missed by both methods. The area under the ROC curve (AUC) of DTTP, catheter tip culture and the combination method was 0.912, 0.882 and 0.941 for diagnosis of CRBSI, respectively. Validity values for the diagnosis of CRBSI for DTTP were: sensitivity 82.35%, specificity 92.31%, positive predictive value 93.33% and negative predictive value 80.00%, and they were higher than those of the catheter tip culture method only (76.47%, 84.62%, 86.67% and 73.33%). The specificity and positive predictive CRBSI combination of the two methods in the diagnosis value were up to 100%, the sensitivity (88.24%) and negative predictive value (86.67%) was also increased, but no significant differences were found with DTTP method (χ2=0.00, P=1.00; χ2=0.00, P=0.98; χ2=0.00, P=0.98; χ2=0.00, P=0.98). DTTP can be a valid method recommended for CRBSI diagnosis in critically ill patients with acceptable sensitivity, good specificity as well as positive predictive value. DTTP combined with other clinical symptoms can not only avoid unnecessary catheter withdrawal, but it also can help obtain the optimal treatment time and strategy.

Transfusion with synthetic platelet substitutes, H12-(ADP)liposomes, effectively rescues thrombocytopenic rabbits from massive hemorrhage

Event Abstract Back to Event Transfusion with synthetic platelet substitutes, H12-(ADP)liposomes, effectively rescues thrombocytopenic rabbits from massive hemorrhage Masato Takikawa1, Kosuke Hagisawa2, Manabu Kinoshita3, Haruka Asahina4, Daizo Saito5 and Shinji Takeoka1 1 Waseda University, Graduate School of Advanced Science and Engineering, Japan 2 National Defense Medical College, Department of Physiology, Japan 3 National Defense Medical College, Department of Immunology and Microbioogy, Japan 4 National Defense Medical College, Department of Traumatology and Critical Care Medicine, Japan 5 National Defense Medical College Research Institute, Division of Traumatology, Japan Introduction: Hemorrhagic shock and the following coagulopathy are critical conditions in traumatic patients[1]. Resuscitative transfusion composed of plasma, red blood cells (RBCs) and platelets (PLTs) ratios of 1:1:1 was reported to be effective in rescuing such fatally injured patients. Although PLT transfusion is desirable to treat the coagulopathy, it is restricted in particular prehospital resuscitation due to its short shelf life. In order to address this crucial issue, we have developed H12-(ADP)liposomes as a liposome-type PLTs substitute[2]. H12-(ADP)liposomes are conjugated with dodecapeptides (H12, HHLGGAKQAGDV), which is a carboxy terminal sequence of fibrinogen gamma-chain, on liposomes’ surface, and encapsulated with adenosine 5’-phosphate (ADP), which enhances PLT aggregation. We investigated a hemostatic effect of H12-(ADP)liposomes on the massive hemorrhage in thrombocytopenic rabbits. Materials and Methods: Sixteen male NZW rabbits weighed 2.4 kg were used. Thrombocytopenia (PLT count; approximately 46×103/μL) was induced in the rabbits by repeated blood withdrawal and isovolemic transfusion of autologous washed RBCs. Thereafter, a liver injury was made followed by balloon compression for 5 min. During balloon compression, H12-(ADP)liposomes/PPP (Platelet-Poor Plasma) (n=6), PRP (Platelet-Rich Plasma) (n=6), PPP (n=4) was transfused to the rabbits. After the removal of compression, bleeding volume from the injured site was measured for the initial 10 min. Bleeding time was also monitored in the subsequent 120 min. After achieving hemostasis, transfusion of RBCs from other rabbits ([Hb]≒17.1 g/dL) was performed to recover from the severe hypotension and hypohemoglobinemia. Thereafter, their survivals were observed for 48 hours. Results: Rabbits in the H12-(ADP)liposomes/PPP transfusion group stopped bleeding at 47 min after liver injury (PRP group stopped at 37 min), and their bleeding volume for 10 min after liver injury was 18.9 g (PRP group; 19.4 g). None of the rabbits in PPP group was able to achieve hemostasis and thereby survived beyond 3 hours. RBCs transfusion after achieving hemostasis raised MAP from 53 to 59 mmHg in H12-(ADP)liposomes/PPP and PRP groups. Survival rates of H12-(ADP)liposomes/PPP and PRP groups were 83%. Discussions: Transfusion with H12-(ADP)liposomes/PPP as well as PRP effectively reduced bleeding time and volume in rabbits comparing to the transfusion with PPP. There were no significant differences in bleeding time/volume between these two groups. Conclusion: Thus, H12-(ADP)liposomes may have a potent hemostatic effect and thereby improve the survival rate that is equivalent to PRP.

Abstract 11934: Resuscitation by Transfusion With Hemoglobin Vesicles in Trauma Hemorrhagic Shock / Coagulopathy Rabbits

Introduction: We have developed Hemoglobin vesicle (HbV) as an artificial substitute for Red blood cells (RBC). Hypothesis: To evaluate the efficacy of HbV for alternative treatment of massive transfusion protocol (MTP) in traumatic hemorrhagic shock / coagulopathy rabbits. Methods: Hemorrhagic shock / thrombocytopenia were induced in rabbits by repeated blood withdrawal (total 400ml) and isovolemic transfusion of autologous washed RBC. Liver penetrating injury led lethal oozing hemorrhage. Balloon compression and Platelet rich plasma (PRP) administration stopped bleeding. Thereafter, allogenic RBC transfusion, HbV or 5% Albumin was administered to the animals (n=30). Acute prognosis was compared among them for 24 hr. Hemodynamic and hematologic parameters were periodically recorded for first 1hr. Results: In anemic and thrombocytopenic rabbits (Hb &lt; 6.0 g/dl, platelets &lt; 40,000 /μL, mean arterial pressure (MAP) &lt; 50 mmHg), liver penetrating injury caused additional bleeding and animals suffered class IV shock (Hb &lt; 5.0 g/dl, MAP &lt; 40 mmHg). PRP administration restored platelets ≧ 70,000 /μL, which stopped bleeding within 40 min. Subsequent administration of HbV as well as RBC transfusion regained MAP more than 50 mmHg, and they rescued 70 % animals from liver hemorrhage, although rabbits receiving 5% albumin showed 10% survival in the first 24 hours. HbV administration did not affect the coagulation parameters as well as RBC transfusion. Conclusions: HbV may be effectively instead of RBCs for acute hemorrhagic shock and trauma induced coagulopathy.

Blood withdrawal affects iron store dynamics in primates with consequences on monoaminergic system function

Iron homeostasis is essential for the integrity of brain monoaminergic functions and its deregulation might be involved in neurological movement disorders such as the restless legs syndrome (RLS). Although iron metabolism breakdown concomitantly appears with monoaminergic system dysfunction in iron-deficient rodents and in RLS patients, the direct consequences of peripheral iron deficiency in the central nervous system (CNS) of non-human primates have received little attention. Here, we evaluated the peripheral iron-depletion impact on brain monoamine levels in macaque monkeys. After documenting circadian variations of iron and iron-related proteins (hemoglobin, ferritin and transferrin) in both serum and cerebrospinal fluid (CSF) of normal macaques, repeated blood withdrawals (RBW) were used to reduce peripheral iron-related parameter levels. Decreased serum iron levels were paradoxically associated with increased CSF iron concentrations. Despite limited consequences on tissue monoamine contents (dopamine – DA, 3, 4-dihydroxyphenylacetic acid – DOPAC, homovanillic acid, L-3, 4-dihydroxyphenylalanine – L-DOPA, 5–8 hydroxytryptamine – 5-HT, 5-hydroxyindoleacetic acid – 5-HIAA and noradrenaline) measured with post-mortem chromatography, we found distinct and region-dependent relationships of these tissue concentrations with CSF iron and/or serum iron and/or blood hemoglobin. Additionally, striatal extracellular DA, DOPAC and 5-HIAA levels evaluated by in vivo microdialysis showed a substantial increase, suggesting an overall increase in both DA and 5-HT tones. Finally, a trending increase in general locomotor activity, measured by actimetry, was observed in the most serum iron-depleted macaques. Taken together, our data are compatible with an increase in nigrostriatal DAergic function in the event of iron deficiency and point to a specific alteration of the 5-HT/DA interaction in the CNS that is possibly involved in the etiology of RLS.

Treatment with fibrinogen γ-chain peptide-coated, adenosine 5'-diphosphate-encapsulated liposomes as an infusible hemostatic agent against active liver bleeding in rabbits with acute thrombocytopenia.

We evaluated the hemostatic efficacy of H12-(adenosine 5'-diphosphate [ADP])-liposomes in the setting of active liver bleeding in rabbits with dilutional thrombocytopenia after massive transfusion. Acute thrombocytopenia (platelet [PLT] count < 50 × 10(9) /L) was induced in rabbits by repeated blood withdrawal and isovolemic transfusion of autologous washed red blood cells. Liver hemorrhage was initiated by a penetrating liver injury. Subsequently, the animals received tamponade treatment for the liver hemorrhage for 5 minutes and were intravenously administered H12-(ADP)-liposomes with PLT-poor plasma (PPP), PLT-rich plasma (PRP), PPP alone, H12-(phosphate-buffered saline [PBS])-liposome/PPP, or H12-(ADP)-liposomes/PPP plus fibrinogen concentrate during the tamponade. Administration of H12-(ADP)-liposomes/PPP rescued 60% of the rabbits from the liver hemorrhage; PRP administration rescued 50%. In contrast, rabbits receiving PPP or H12-(PBS)-liposome/PPP achieved only 10 or 17% survival, respectively, for the first 24 hours. H12-(ADP)-liposomes/PPP as well as PRP consistently reduced bleeding volumes and shortened clotting times (CTs) in comparison to PPP administration. Specifically, bleeding volumes in the initial 5 minutes averaged 11 mL (H12-(ADP)-liposomes/PPP) and 17 mL (PRP) versus 30 mL (PPP; p < 0.05); CTs averaged 270 and 306 seconds versus 401 seconds (p < 0.05). H12-(ADP)-liposomes were observed at the bleeding site with thrombus formation, suggesting an induction of thrombi. Neither macro- nor microthrombi were detected in the lung, kidney, spleen, or liver in rabbits treated with H12-(ADP)-liposomes. Supplementation of fibrinogen to H12-(ADP)-liposomes/PPP did not significantly improve rabbit survival. H12-(ADP)-liposomes might be a safe and effective therapeutic tool during damage control surgery for trauma patients with acute thrombocytopenia and massive bleeding.

Interscapular site for transcutaneous bilirubin measurement in preterm infants: a better and safer screening site

To prevent bilirubin induced brain injury in the newborn, repeated blood withdrawals are necessary to ascertain bilirubin levels and institute care. Noninvasive, painless and bloodless screening using transcutaneous bilirubinometry is the standard of care for term and near term neonates but there is still debate about its use with preterm neonates. The aims of this study were to determine how transcutaneous bilirubin (TcB) level measured from the interscapular site related to total serum bilirubin (TSB) level and to compare performance of TcB from the forehead, sternum and interscapular sites in identifying preterm neonates in need of phototherapy. This was a cross sectional study conducted at Groote Schuur level III neonatal unit. Over a 5-month period 122 consecutive preterm neonates <35 weeks gestational age were enrolled. TcBs were measured over the forehead, sternum and interscapular area. Pearson's correlation coefficients and differences between TSB and TcBs were computed. P-value <0.05 was considered significant. The median gestational age of the study participants was 31 weeks (range: 24 to 34 weeks), the median TSB level was 81.5 μmol l(-1) (range: 25 to 229 μmol l(-1)) and 45% had a TSB at the phototherapy threshold. The correlation coefficients for TcBs ranged from 0.859 to 0.929 (P<0.001). The difference between TSB and TcBs ranged from -86 to +51 μmol l(-1). With respect to initiating phototherapy, the interscapular site had the highest sensitivity of 94% and lowest false negative rate of 6%. Using transcutaneous bilirubinometry, the interscapular site is superior and safer for screening preterm neonates.

Abstract 357: Treatment With Fibrinogen ?-chain Peptide-coated, Adenosine Diphosphate-encapsulated Liposomes as an Infusible Haemostatic Agent for Dilutional Coagulopathy

Background: We developed a fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV, H12)-coated, adenosine-diphosphate (ADP)-encapsulated liposomes [H12-(ADP)-liposomes] that accumulate at bleeding site via interaction with activated platelets via GPIIb/IIIa and augment platelet aggregation by releasing ADP. Objective: To evaluate the haemostatic efficacy of H12-(ADP)-liposomes against active liver bleeding in dilutional thrombocytopenic rabbits following massive transfusion. Methods: Acute thrombocytopenia (platelets &lt; 50 х 10 3 /μL) was induced in rabbits by repeated blood withdrawal and isovolemic transfusion of autologous washed red blood cells. In these thrombocytopenic rabbits, liver hemorrhage was made by penetrating liver injury. Subsequently, they received the astriction treatment against the liver hemorrhage for 5 min and were intravenously administered with H12-(ADP)-liposomes with platelet-poor plasma (PPP), platelet-rich plasma (PRP), PPP alone, H12-(PBS)-liposome/PPP or H12-(ADP)-liposomes/PPP + fibrinogen concentrate during the astriction. Results: Administration of H12-(ADP)-liposomes as well as PRP rescued 60% rabbits from the liver hemorrhage (PRP, 50%), although rabbits receiving PPP or H12-(PBS)-liposome/PPP showed 10 or 17% survival in the first 24 h, respectively. Administration of H12-(ADP)-liposomes as well as PRP potently reduced both bleeding volume and time from the liver injury site. H12-(ADP)-liposomes were observed at the bleeding site in the liver with thrombus formation, suggesting an induction of thrombi. Whereas, neither macro- nor micro-thrombi were detected in the lung, kidney or liver in rabbits treated with H12-(ADP)-liposomes. Supplementation of fibrinogen to H12-(ADP)-liposomes/PPP did not significantly improve rabbit survivals (40% survival). Conclusions: H12-(ADP)-liposomes may be a safe and effective therapeutic tool during damage control surgery for acute thrombocytopenic trauma patients with massive bleeding.

Preload‐adjusted left ventricular dP/dtmax: a sensitive, continuous, load‐independent contractility index

The classical indicators of left ventricular (LV) performances have been derived from pressure-volume (PV) and stroke work-volume plots obtained during various loading or pharmacological interventions. More recently, the preload-adjusted maximal change in pressure over time (PAdP/dtmax), derived from single beat PV analysis, has been shown to reflect the LV systolic performance accurately in varying conditions of inotropy and afterload. The objective of this study was to address whether PAdP/dtmax is a valid indicator of LV function in the setting of large preload variations, compared with the classical dP/dtmax-end-diastolic volume (EDV) and stroke work-EDV (preload recruitable stroke work) relationships. Nine anaesthetized and mechanically ventilated rats were instrumented with a ventricular conductance catheter. Stepwise preload reduction was achieved by repeated blood withdrawals (up to a total of 5 ml). Steady-state and dynamic PV loops were recorded during brief occlusion of the inferior vena cava, and LV function parameters were derived from these recordings. Our results demonstrate that PAdP/dtmax behaved in a similar manner to preload recruitable stroke work, reflecting well-maintained LV contractility during controlled haemorrhage until mean arterial pressure decreased below 40 mmHg. In contrast, dP/dtmax-EDV increased significantly and exhibited a curvilinear response that was associated with a large inter- and intra-animal variability. In a model of acute preload reduction, PAdP/dtmax was found to be the best indicator of systolic LV function. Given its simplicity, this real-time index derived from single beat analysis should be tested further in clinical settings.

Fibrinogen γ‐chain peptide‐coated, ADP‐encapsulated liposomes rescue thrombocytopenic rabbits from non‐compressible liver hemorrhage

We developed a fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV [H12])-coated, ADP-encapsulated liposome (H12-[ADP]-liposome) that accumulates at bleeding sites via interaction with activated platelets via glycoprotein IIb-IIIa and augments platelet aggregation by releasing ADP. To evaluate the efficacy of H12-(ADP)-liposomes for treating liver hemorrhage in rabbits with acute thrombocytopenia. Thrombocytopenia (platelets < 50 000 μL(-1)) was induced in rabbits by repeated blood withdrawal (100 mL kg(-1) in total) and isovolemic transfusion of autologous washed red blood cells. H12-(ADP)-liposomes with platelet-poor plasma (PPP), platelet-rich plasma (PRP), PPP, ADP liposomes with PPP or H12-(PBS)-liposomes/PPP, were administered to the thrombocytopenic rabbits, and liver hemorrhage was induced by penetrating liver injury. Administration of H12-(ADP)-liposomes and of PRP rescued all thrombocytopenic rabbits from liver hemorrhage as a result of potent hemostasis at the liver bleeding site, although rabbits receiving PPP or ADP liposomes showed 20% survival in the first 24 h. Administration of H12-(ADP)-liposomes and of PRP suppressed both bleeding volume and time from the site of liver injury. H12-(phosphate-buffered saline)-liposomes lacking ADP also improved rabbit survival after liver hemorrhage, although their hemostatic effect was weaker. In rabbits with severe thrombocytopenia (25 000 platelets μL(-1)), the hemostatic effects of H12-(ADP)-liposomes tended to be attenuated as compared with those of PRP treatment. Histologic examination revealed that H12-(ADP)-liposomes accumulated at the bleeding site in the liver. Notably, neither macrothombi nor microthrombi were detected in the lung, kidney or liver in rabbits treated with H12-(ADP)-liposomes. H12-(ADP)-liposomes appear to be a safe and effective therapeutic tool for acute thrombocytopenic trauma patients with massive bleeding.

Angiotensin II-dependent hypertension causes reversible changes in the platelet proteome

Hypertension is a risk factor for arterial thrombosis. We investigated the effects of angiotensin II (ANG II)-dependent hypertension on the platelet proteome. Hypertension was induced in cyp1a1ren-2 transgenic rats by feeding indole-3-carbinol (n = 10) and in Fischer 344 rats by subcutaneously infusing ANG II (n = 7). After 14 days of hypertension (SBP 180 mmHg) and 10 days after normalization of blood pressure, changes in the platelet proteome were assessed by two-dimensional differential in-gel electrophoresis. In a subset of animals (n = 4), repeated blood withdrawals were performed. Of 1040 protein spots, 45 displayed hypertension-associated changes (>1.5-fold, P < 0.01) in both models (34 increased, 11 decreased). All were reversible within 10 days. Thirty-eight spots were identified by mass spectrometry and assigned to 20 distinct proteins. The majority of spots with increased intensity constituted protein fragments. Repeated blood withdrawals and stimulation of megakaryocytopoiesis by a thrombopoietin receptor agonist induced changes in the same protein spots but in the opposite direction to those induced by ANG II-dependent hypertension. ANG II-dependent hypertension is associated with enhanced protein degradation in platelets. As these changes are reversible, the proteins identified might be used to develop a biomarker for monitoring recent blood pressure history.

Blood flow index using near-infrared spectroscopy and indocyanine green as a minimally invasive tool to assess respiratory muscle blood flow in humans

Near-infrared spectroscopy (NIRS) in combination with indocyanine green (ICG) dye has recently been used to measure respiratory muscle blood flow (RMBF) in humans. This method is based on the Fick principle and is determined by measuring ICG in the respiratory muscles using transcutaneous NIRS in relation to the [ICG] in arterial blood as measured using photodensitometry. This method is invasive since it requires arterial cannulation, repeated blood withdrawals, and reinfusions. A less invasive alternative is to calculate a relative measure of blood flow known as the blood flow index (BFI), which is based solely on the NIRS ICG curve, thus negating the need for arterial cannulation. Accordingly, the purpose of this study was to determine whether BFI can be used to measure RMBF at rest and during voluntary isocapnic hyperpnea at 25, 40, 55, and 70% of maximal voluntary ventilation in seven healthy humans. BFI was calculated as the change in maximal [ICG] divided by the rise time of the NIRS-derived ICG curve. Intercostal and sternocleidomastoid muscle BFI were correlated with simultaneously measured work of breathing and electromyography (EMG) data from the same muscles. BFI showed strong relationships with the work of breathing and EMG for both respiratory muscles. The coefficients of determination (R(2)) comparing BFI vs. the work of breathing for the intercostal and sternocleidomastoid muscles were 0.887 (P < 0.001) and 0.863 (P < 0.001), respectively, whereas the R(2) for BFI vs. EMG for the intercostal and sternocleidomastoid muscles were 0.879 (P < 0.001) and 0.930 (P < 0.001), respectively. These data suggest that the BFI closely reflects RMBF in conscious humans across a wide range of ventilations and provides a less invasive and less technically demanding alternative to measuring RMBF.

The bone metabolism, repeated blood withdrawals and iron enriched diet in rats

The bone metabolism, repeated blood withdrawals and iron enriched diet in rats

Erythropoietin Injections Stimulate Spleen BMP4-Dependant Stress Erythropoiesis in a Mouse Model of Generalized Inflammation.

Abstract 1983Poster Board I-1005 Introduction:In mouse, acute anemia leads to the rapid expansion and differentiation of stress erythroid progenitors in the spleen. It has been shown that these progenitors respond to BMP4, Stem Cell Factor and hypoxia and differentiate into stress BFU-E. These are sensitive to high levels of erythropoietin (EPO) and rapidly expand in the spleen, allowing rapid recovery from the anemia (JM Perry et al., Blood 2009, 113:911-918). Inflammation is known to inhibit growth and differentiation of erythroid progenitors and to suppress EPO synthesis in the kidney. However, the effect of pro-inflammatory cytokines on this stress erythropoiesis response is not known. We have recently developed a mouse model of zymosan-induced generalized inflammation and shown that stimulation of erythropoiesis by repeated blood withdrawal or injections of erythropoietin favours iron mobilization from tissue iron stores (S. Lasocki et al., CCM 2008, 36:2388-2394), suggesting that EPO treatment may be beneficial provided effective erythropoiesis can be elicited. Objectives:The aim of our study was to assess the impact of EPO injections on the stress erythropoietic response in this mouse model of chronic inflammation. Methods:Mice (C57BL/6) received a single intraperitoneal injection of zymosan at day 1 (Z1) followed by four consecutive daily injections of EPO at day 5, 6, 7 and 8. Mice were analyzed one day (Z9EPO1), four days (Z12EPO4) or nine days (Z17EPO9) after the final injection and compared to controls, Z alone or EPO alone. Double Ter119/CD71 labelling was used to analyze the different stages of erythroblast differentiation by FACS, in bone marrow and spleen in the different conditions. Spleen BMP4 expression was followed by RT-qPCR and immunohistochemistry. Serum EPO levels were measured by ELISA and haematological parameters were recorded. Results:In the inflammatory condition, bone marrow erythropoiesis is suppressed and does not respond to EPO injections. There is a concomitant increased in the percentage of apoptotic Ter119+ cells. In the spleen, inflammation increases spleen size but only moderately stimulates the percentage of erythroblasts. However, EPO injections lead to a 10-fold increase in the percentage of immature erythroblasts at Z9EPO1, followed three days later (Z12EPO4) by a similar increase in the proportion of mature erythroblasts. This finally results in increased reticulocytes and haemoglobin concentration.In the spleen, BMP4 mRNAs are not stimulated by inflammation but significantly increased by EPO injections, both in normal mice and mice with Zymosan-induced inflammation. The protein BMP4 is expressed by erythroid precursors and stromal cells. Double labelling with F4/80 and BMP4 clearly shows that spleen macrophages are the BMP4-expressing cells following EPO injections in mice with a generalized inflammation. Conclusion:In mouse, bone marrow erythropoiesis is repressed by inflammation as it has been shown for human erythropoiesis and it does not respond to EPO injections. By contrast, spleen stress erythropoiesis is strongly stimulated by injections of EPO despite the presence of inflammation. This results from a strong increase in BMP4 synthesis by spleen macrophages. BMP4 is known to be stimulated by acute anemia but our study is the first report of a direct effect of EPO injections on BMP4 expression in the spleen and of the identification of macrophages as the stromal cells producing BMP4.It will be of interest to find out if bone marrow macrophages in humans can synthesize BMP4 and also contribute to a medullar stress erythropoietic response. Disclosures:No relevant conflicts of interest to declare.

Iron enriched diet, repeated blood withdrawals and their effect on bone metabolism in rats

CT scan (Siemens 64) using a fixed threshold of 450 mg/cm3 and Mindways Software (BIT-2) to analyse both hips (1mm slice thickness, 0.59mmvoxel size). The starting position for cross sectionswas a 1mm thickmid-femoral neck slice at an eccentricity of 1.4, since this location along the neck axis was reproducible and unaffected by age (mean 51%, SE 0.016%). 5 parallel 1 mm slices were evaluated towards the midline. Age effects on trBMD (mg/cm3) were evaluated using repeated measures MANOVA. TrBMD was significantly lower at 85 than at 25 years old amounting to over 50% difference in 3 out of 4 quadrants (Table 1). However, in the infero-anterior quadrants (that receive maximal stress at the heel strike in normal gait) there was very little association with age (r only 0.094) and only a 24% difference between 85 and 25 (MANOVA p<0.0001). Body weight had no association with trabecular BMD in any quadrant (unlike cortical thickness). Trabecular bone was better preserved in the infero-anterior quadrant in elderly women than in the other 3 quadrants. Since tbBMD also makes a significant contribution to failure load of femoral necks ex-vivo, increasing trabecular bone in the remaining quadrants should be included alongside efforts to strengthen the cortex in strategies to prevent hip fracture.

Evaluating tissue perfusion using labelled water indicator microdialysis in a rat model of haemorrhagic shock

The indicator microdialysis technique with 3H2O was evaluated as a method for assessing local blood flow in skeletal muscle and liver in a rat model of haemorrhagic shock. Microdialysis probes were inserted into the hind limb muscle and liver of 12 rats and perfused with a solution containing 3H2O. Haemorrhagic shock was induced in eight rats by repeated blood withdrawals; four rats were used as a control group. The outflow to inflow (O/I) ratio of water activity, lactate and pyruvate contents in the dialysate were repeatedly measured. The ratio of lactate to pyruvate (L/P) was calculated. There was no correlation between blood loss and O/I ratio (r = 0.323 for liver, r = 0.300 for muscle), between the mean arterial pressure and O/I ratio (r = 0.460 for liver, r = −0.301 for muscle) or between the concentration of lactate and O/I ratio (r = −0.237 for liver, r = 0.454 for muscle). A significant correlation was found between blood loss and lactate concentration in muscle (r = 0.619, p < 0.0001). We suppose that microdialysis with 3H2O cannot be used as a sensitive method to estimate regional blood flow changes during haemorrhagic shock in rats. Measuring lactate, pyruvate and L/P ratio in the microdialysate seems to be a superior method to assess tissue hypoperfusion caused by haemorrhagic shock in rats.