Platelets loaded with liposome-encapsulated thrombin have increased coagulability.

Essentials Platelet transfusions can have limited efficacy during hemorrhage associated with coagulopathy. Thrombin can be shielded by encapsulation into nanoliposomes and delivered to platelets ex vivo. Loading platelets with liposomal thrombin improved several aspects of platelet coagulability. Platelets loaded with liposomal thrombin can overcome some coagulopathic deficiencies in vitro. SUMMARY: Background Platelets are integral to clot formation and are often transfused to stop or prevent bleeding. However, transfusions of platelets are not always effective, particularly in the most severe cases of hemorrhage. Nanoparticle systems have been developed to mimic platelets but inherently lack important aspects of platelet function, which limits their potential effectiveness. Objectives Increasing the natural coagulability of transfusable platelets could increase their efficacy during treatment of severe hemorrhage. Thrombin is a potent platelet agonist that currently cannot be used intravenously because of the risk of thrombosis. We hypothesized that delivery of thrombin to ex vivo platelets via liposomal encapsulation would enable transfusable platelets to become more coagulable in response to platelet agonists. Methods Thrombin was encapsulated into nanoliposomes and delivered to platelets ex vivo. Platelet coagulability was measured by monitoring platelet activation, clot contraction, clot time and clot stability in several in vitro assays. These parameters were also measured under conditions where coagulation is compromised, including during acidosis, antiplatelet drugs, hemophilia A and trauma-induced coagulopathy. Results Liposomal thrombin was endocytosed and used by platelets ex vivo but was not secreted upon activation. These modified platelets became more sensitive and responsive to agonists and improved clotting time even under conditions that normally cause platelet dysfunction or have impaired coagulation. Conclusions Several aspects of platelet function were enhanced by ex vivo delivery of liposomal thrombin.

Rediscovering the wound hematoma as a site of hemostasis during major arterial hemorrhage.

BACKGROUND: Treatments for major internal bleeding after injury include permissive hypotension to decrease the rate of blood loss, intravenous infusion of plasma or clotting factors to improve clot formation, and rapid surgical hemostasis or arterial embolization to control bleeding vessels. Yet, little is known regarding major internal arterial hemostasis, or how these commonly used treatments might influence hemostasis. OBJECTIVES: (i) To use a swine model of femoral artery bleeding to understand the perivascular hemostatic response to contained arterial hemorrhage. (ii) To directly confirm the association between hemodynamics and bleeding velocity. (iii) To observe the feasibility of delivering an activated clotting factor directly to internal sites of bleeding using a simplified angiographic approach. METHODS: Ultrasound was used to measure bleeding velocity and in vivo clot formation by elastography in a swine model of contained femoral artery bleeding with fluid resuscitation. A swine model of internal pelvic and axillary artery hemorrhage was also used to demonstrate the feasibility of local delivery of an activated clotting factor. RESULTS: In this model, clots formed slowly within the peri-wound hematoma, but eventually contained the bleeding. Central hemodynamics correlated positively with bleeding velocity. Infusion of recombinant human activated factor VII into the injured artery near the site of major internal hemorrhage in the pelvis and axillae was feasible. CONCLUSIONS: We rediscovered that clot formation within the peri-wound hematoma is an integral component of hemostasis and a feasible target for the treatment of major internal bleeding using activated clotting factors delivered using a simplified angiographic approach.

A call for a value based approach to laboratory medicine funding.

All areas of healthcare, including pathology, are being challenged by the reality that the days of ever increasing budgets are over and the key debate is about how to provide value for money. As originally described by Porter and Tiesberg, value-based healthcare is defined as maximising outcomes over cost by moving away from fee for service models to ones that reward providers on the basis of outcomes (1). While production efficiencies will continue to evolve, the opportunities for future stepwise improvements in production costs are likely to have diminished. The focus now is on delivering improved testing outcomes in a relatively cost neutral or at least cost effective way. This brings pathology into line with other health services that focus on value for money for payers, and maximising health outcomes for consumers. This would signal a break from the existing pathology funding model, which does not directly recognise or reward the contribution of pathology towards improved health outcomes, or seek to decommission tests that offer little clinical value. Pathology has a direct impact on clinical and economic outcomes that extend from testing and it is important to garner support for a new approach to funding that incentivises improvements of the overall quality and contribution of the pathology service.

HIV evolution: CTL escape mutation and reversion after transmission.

Within-patient HIV evolution reflects the strong selection pressure driving viral escape from cytotoxic T-lymphocyte (CTL) recognition. Whether this intrapatient accumulation of escape mutations translates into HIV evolution at the population level has not been evaluated. We studied over 300 patients drawn from the B- and C-clade epidemics, focusing on human leukocyte antigen (HLA) alleles HLA-B57 and HLA-B5801, which are associated with long-term HIV control and are therefore likely to exert strong selection pressure on the virus. The CTL response dominating acute infection in HLA-B57/5801-positive subjects drove positive selection of an escape mutation that reverted to wild-type after transmission to HLA-B57/5801-negative individuals. A second escape mutation within the epitope, by contrast, was maintained after transmission. These data show that the process of accumulation of escape mutations within HIV is not inevitable. Complex epitope- and residue-specific selection forces, including CTL-mediated positive selection pressure and virus-mediated purifying selection, operate in tandem to shape HIV evolution at the population level.

The value of blood lactate measurements in ICU: an evaluation of the role in the management of patients on haemofiltration.

In response to clinical demand some point-of-care analysers now provide blood lactate measurements, but recently concern has been expressed about the value and interpretation of these measurements. We undertook this study to evaluate blood lactate measurements in patients with acute renal failure undergoing haemofiltration (HF) with lactate replacement fluid. At baseline, 27 patients had base deficits of >5 mmol/l and 14 (52%) had blood lactates of >3.5 mmol/l. Lactate 'tolerance' was monitored by peak changes in these parameters during the procedure. There was a worsening of base deficit in only three of the patients in whom lactate rises exceeded 10 mmol/l with one survivor. Twelve patients with rises of blood lactate greater than 5 mmol/l improved their base deficit (+1 to +17) with eight (67%) survivors. Of the remaining 12 patients with improved base deficit (+2 to +20), 10 (83%) survived. Lactate tolerance was compromised in patients with co-incidental liver disease, those on inotropic support, and in patients with initial blood lactate measurements of >10 mmol/l and large base deficits. The data suggest that blood lactate and simultaneous acid-base response measurements during HF help to assign correct buffer replacement and should be performed on all patients.

A connectivity standard for point-of-care testing.

A software standard has been designed that will facilitate the linking of all point-of-care testing (POCT) devices to information systems. This standard will replace the myriad of proprietary and incompatible solutions that currently exist. The standard is expected to be a feature of new POCT instruments by 2002.

Purification and analysis of expression of the stationary phase-inducible slp lipoprotein in Escherichia coli: role of the Mar system.

Entrance into the stationary phase in Escherichia coli induces a group of stress response genes including slp, which encodes an outer membrane lipoprotein. Glucose limitation is sufficient, but not necessary, for the increase in slp expression. The Slp protein was purified and an antibody-based assay was developed, which enabled identification of Slp as spot R2226 (G018.1) in the E. coli gene-protein database. Although Slp is a major membrane component in stationary phase cultures grown in complex medium, no significant changes in resistance to oxidative stress or membrane perturbants were found in a slp null mutant strain. The presence or absence of multiple antibiotic resistance (Mar) A did not alter the final stationary phase levels of Slp. However, the Mar system could modestly influence the level of slp expression during the transition from exponential to stationary growth phase. Accumulation of Slp leads to a small increase in sensitivity to chloramphenicol.

Regulated gene expression in Staphylococcus aureus for identifying conditional lethal phenotypes and antibiotic mode of action.

Selectively regulating gene expression in bacteria has provided an important tool for studying gene function. However, well-regulated gene control systems have been restricted primarily for use in laboratory non-pathogenic strains of bacteria (e.g. Escherichia coli, Bacillus subtilis). The development of analogous systems for use in bacterial pathogens such as Staphylococcus aureus would significantly enhance our ability to examine the contribution of any given gene product to pathogen growth and viability. In this report, we adapt, examine and compare three regulated gene expression systems in S. aureus, which had previously been used in B. subtilis. We demonstrate that all three systems function and exhibit titratable induction, together covering a dynamic range of gene expression of approximately 3000-fold. This dynamic range correlates well with the physiological expression levels of cellular proteins. Importantly, we show that one of these systems, the Spac system, is particularly useful for examining gene essentiality and creating specific conditional lethal phenotypes. Moreover, we find that titration of selective target gene products using this system allows direct demonstration of antibiotic mode of action.

The Value of Blood Lactate Measurements in ICU: An Evaluation of the Role in the Management of Patients on Haemofiltration.

In response to clinical demand some point-of-care analysers now provide blood lactate measurements in critical care. Recent literature has raised concerns about the value and interpretation of these measurements. Two particular concerns relate to over-interpretation of lactate rises as equating tissue hypoxia and also the failure to recognise the contribution from inotropic support. We undertook this study to evaluate blood lactate measurements in intensive care unit (ICU) patients in the assessment of response to, and requirements for, haemofiltration (HF) with lactate replacement fluid and to evaluate influences from hepatic failure and from inotropic supportive therapy. Haemofiltration is a convenient renal replacement therapy widely used in intensive care management as an alternative to haemodialysis. Mainly used for the treatment of acute renal failure the process involves removal by filtration of fluid, electrolytes, metabolites and other substances and simultaneous replacement of essential fluid and electrolytes as well as a buffer, usually in the form of lactate (sodium salt). There is controversy about whether lactate replacement may be harmful to the patient and, if so, when it would be appropriate to use a lactate-free fluid at greater expense. Serial blood lactate with simultaneous blood gas measurements were recorded in 27 patients requiring HF for acute renal failure. At baseline all patients had base deficits of >5mmol/L and 14 (52%) had blood lactates of >3.5mmol/L. Lactate 'tolerance' was monitored by peak changes in these parameters during the procedure. There was a worsening of base deficit in only three of the patients in whom lactate rises exceeded 10 mmol/L at some stage during HF with one survivor. A further twelve patients with rises of blood lactate greater than 5 mmol/L improved their base deficit (+1 to +17) with 8 (67%) survivors. Of the remaining twelve patients with improved base deficit (+2 to +20), 10(83%) survived. The influence on 'lactate tolerance' in patients with co-incidental liver disease and those on inotropic support was studied. In these groups lactate tolerance was compromised, particularly those on adrenaline support. Patients with initial blood lactate measurements of >10mmol/l and large base deficits were also lactate intolerant. The data suggest that rises in blood lactate during HF signal harm if accompanied by inadequate improvement in base deficit. Blood lactate and simultaneous acid-base response measurements during HF help to assign correct buffer replacement and should be performed on all patients.

Connectivity: the key to point-of-care testing.

For the point-of-care testing market to grow successfully the advances in whole blood sensing technology must be accompanied by improvements in information technology to link the analytical devices to data management systems. A common connectivity standard for all instruments is being developed to replace the myriad of proprietary and incompatible solutions that currently exist.

Secondary hyperparathyroidism in patients from Western Australia with hip fracture: relationship to type of hip fracture, renal function, and vitamin D deficiency.

OBJECTIVES: To determine the frequency of vitamin D deficiency and secondary hyperparathyroidism in Australian hip fracture patients living in the community. PATIENTS: A total of 283 consecutive patients with hip fracture admitted over a 15-month period to a university teaching hospital in Western Australia. Included were residents of hostels for the elderly, and excluded were nursing home residents and those with malignant fractures. METHOD: Data collected included biochemistry (25 hydroxyvitamin D, parathyroid hormone and creatinine levels), measurements of function and disability (Barthel Index, Frenchay Activity Index), sunshine exposure, and basic demographics. RESULTS: Vitamin D deficiency occurred in 31.7% and secondary hyperparathyroidism occurred in 17.7% of cases. The major determinants of vitamin D deficiency were outdoor sunshine exposure, ambient daily sunshine, and disability (low Frenchay Activity Index or ADL difficulty). Secondary hyperparathyroidism was related to older age, renal dysfunction, and vitamin D deficiency. Secondary hyperparathyroidism was associated with an excess of trochanteric over subcapital hip fractures. CONCLUSIONS: Secondary hyperparathyroidism appears to be a heterogeneous condition, caused in approximately equal proportions by vitamin D deficiency and renal dysfunction, that may confer increased cortical bone fragility and trochanteric fractures. Renal dysfunction in old age may be an important additional determinant of senile osteoporosis, which has implications for preventive therapy. Vitamin D deficiency occurs in disabled and, presumably, housebound older people despite near optimal climatic conditions.

The effect of estrogen deficiency on bone mineral density, renal calcium and phosphorus handling and calcitropic hormones in the rat.

The oophorectomized (OOX) rat has been proposed as a good model of postmenopausal osteoporosis in women. The aim of this study was to compare the effect of OOX in 6-month-old rats to the effects of menopause in women with respect to bone mass, the renal handling of calcium and phosphorus, and calcitropic hormones. To more closely replicate the human situation the rats were pair fed a 0.1% calcium diet. Thirty four, 6-month-old rats were randomized to sham operation or OOX. Whole body and regional bone density was performed at baseline and 6 weeks postoperation. Blood and 24-hour urine samples were obtained at baseline, 1, 3, and 6 weeks and assayed for various biochemical variables, parathyroid hormone (PTH), and calcitriol. The OOX rats lost significantly more bone than the sham-operated rats (change in global bone mineral density, sham -1.7 +/- 2.0%, OOX -3.9 +/- 2.6%, P < 0.001). In the OOX animals, an increase in the 24-hour urine calcium was observed at 1 and 3 weeks, which had returned to sham-operated levels by 6 weeks. In the whole group, the increase in urine calcium at 1 week was negatively correlated with the change in bone mass at 6 weeks (r = -0.39, P = 0. 029). OOX resulted in an increased filtered load of calcium and phosphorus. There was an increase in the maximal renal tubular reabsorption of phosphorus (TmP-GFR) but no clear change in renal calcium handling. Neither calcitriol nor parathyroid hormone showed a significant change as a result of OOX. As in postmenopausal women, following oophorectomy in the rat, there was significant generalized bone loss and a negative calcium balance. This was associated with an initial rise in urine calcium due to a rise in the filtered calcium load; plasma phosphorus and TmP-GFR also rose. The rat model may differ from postmenopausal bone loss in that the initial rise in urine calcium was not present at later time points as occurs in natural menopause in women. Calcitropic hormone levels did not change. This study has shown that the 6-month-old OOX rat fed a 0.1% calcium diet has many similarities of calcium and phosphorus homeostasis to that seen at menopause in women.

The metabolism of platelet-activating factor in severe and cerebral malaria.

In order to examine the effects of platelet-activating factor (PAF) in complicated Plasmodium falciparum infections, plasma concentrations of lyso-PAF, stable metabolite and principal precursor of PAF, were measured in 25 Vietnamese adults with severe malaria. The concentration of PAF in the cerebrospinal fluid (CSF) was determined in a sub-group of 23 comatose patients and, together with that of lyso-PAF, in the plasma of 20 patients on recovery of consciousness. The concentration of lyso-PAF in the plasma was depressed on admission to hospital (median [range]; 21 [8-143] vs. 293 [215-410] ng/ml in 10 controls; P < 0.001). There was, however, no change in plasma activity of acetylhydrolase which converts PAF to lyso-PAF (P > 0.01 vs. controls) while simultaneous reduction in the concentration of lipoproteins associated with lyso-PAF were less than those of lyso-PAF per se in the plasma. The plasma concentration of lyso-PAF on admission was associated with parasitaemia and the concentration of serum triglycerides (rs = -0.42, P = 0.04 in each case), the latter being consistent with hepatic effects of PAF reported in previous studies. CSF concentrations of PAF on admission were low (2.3 [0.5-7.7] vs. 0.9 [0-2.5] ng/ml after recovery, P < 0.01) compared with values reported previously in bacterial meningitis. Plasma concentrations of lyso-PAF after recovery lay between admission and control values. While increased availability of PAF may reflect parasite burden and may modulate liver-mediated metabolic disturbances such as hypoglycaemia and lactic acidosis, the role of PAF in cerebral malaria is uncertain.

Mineral homoeostasis in acute renal failure complicating severe falciparum malaria.

Disturbances in calcium metabolism in acute renal failure (ARF) remain incompletely understood. Most data are from patients with rhabdomyolysis. As renal impairment commonly accompanies severe malaria in the absence of rhabdomyolysis, falciparum malaria provides an alternative model of mineral homoeostasis in ARF. We studied 25 Vietnamese subjects, aged 18-63 yr, with severe malaria and 10 controls. Fourteen patients had a serum creatinine level of 250 mumol/L or less during treatment (group 1), five developed ARF but were not dialyzed (group 2a), and six required dialysis (group 2b). Group 1 patients presented with mild hypocalcemia (mean +/- SD serum ionized calcium, 1.18 +/- 0.05 vs. 1.23 +/- 0.02 mmol/L in controls; P = 0.01) that persisted until discharge in the presence of normal serum phosphate, PTH, and vitamin D metabolite levels. Group 2 patients were more hypocalcemic on admission (1.10 +/- 0.08 mmol/L; P < 0.0001 vs. controls), especially those in group 2b whose serum ionized calcium fell to 0.88 +/- 0.13 mmol/L when renal dysfunction was maximal. In group 2 patients, the admission serum PTH level was raised (5.4 +/- 3.8 vs. 2.7 +/- 0.9 pmol/L in controls; P < 0.02) and changed reciprocally with calcemia. Significant rises in serum phosphate occurred only in group 2b patients who had depressed serum free 1,25-dihydroxyvitamin D levels throughout. Hypercalcemia did not accompany the diuretic phase of ARF. These data suggest that parathyroid gland dysfunction is a cause of hypocalcemia in severe malaria without ARF, as seen in group 1 patients; in patients with ARF, the effect of the combination of phosphate retention and altered vitamin D metabolism on skeletal PTH sensitivity is of prime significance.

Dynamic assessment of the electrocardiographic QT interval during citrate infusion in healthy volunteers.

OBJECTIVE: To investigate changes in the electrocardiographic QT interval during rapidly induced, sustained hypocalcaemia in healthy volunteers. DESIGN: Serial rate corrected QT measurements were made during and after a variable rate trisodium citrate infusion designed to "clamp" the whole blood ionised calcium concentration 0.20 mmol/l below baseline for 120 min. SUBJECTS: 12 healthy teetotallers aged 19- 36 years who were not receiving medication known to influence calcium homoeostasis. MAIN OUTCOME MEASURES: Whole blood ionised calcium concentration and QaTc intervals (onset of the Q wave to T wave apex divided by the square root of the RR interval). RESULTS: Mean (SD) ionised calcium concentration decreased from 1.18 (0.03) mmol/l preinfusion to values close to target (0.98 mmol/l) between 10 and 120 min. The QaTc interval lengthened from a baseline of 0.309 (0.021) to a maximum 0.343 (0.024) s0.5 at 10 min before returning to a stable level from 15 to 120 min (0.334 (0.023) and 0.330 (0.023) s0.5 respectively). The change from baseline of both variables expressed as a ratio (delta QaTc/ delta [Ca2+]) was greater during rapid induction of hypocalcaemia (at 5 and 10 min) than at other times during and after the infusion (P < 0.02). CONCLUSIONS: The disproportionate prolongation of QaTc interval during prompt induction of hypocalcaemia suggests rate dependency which can be represented by a hysteresis relation between (ionised calcium, QaTc) coordinates. This finding may have clinical implications.

The effects of menopause and age on calcitropic hormones: a cross-sectional study of 655 healthy women aged 35 to 90.

Although women lose 30% of their skeletal mass after the menopause, the mechanism of this loss is uncertain. Clearly estrogen deficiency is important but whether this works only through direct effects on the skeleton is uncertain. To examine these mechanisms further we have evaluated calcium-related metabolic factors in 655 healthy women. Fasting blood samples were collected from all subjects who were up to 35 years past the menopause, and fasting urine and 24-h urine samples were collected in 365 women who were up to 25 years past the menopause. In the first 15 years postmenopause, there was a rise in total plasma calcium due to a rise in albumin. Bone resorption (hydroxyproline creatinine ratio), bone formation (alkaline phosphatase), and the urine calcium creatinine ratio all rose at menopause and remained elevated for the next 25 years. There was a transient further rise in bone resorption for the 10 years following menopause. Neither PTH nor the free calcitriol index changed for the first 10 years following menopause. Ten years past the menopause, although total calcitriol rose, the free calcitriol index fell due to a rise in vitamin D binding protein. PTH began to rise at 15 years past menopause. GFR fell gradually over the 25 years following menopause. Thus following menopause there is an increase in bone turnover and increased urine calcium loss independent of any effect of PTH or calcitriol, suggesting a direct effect of estrogen deficiency on bone and kidney.(ABSTRACT TRUNCATED AT 250 WORDS)