Rapid and specific detection of clinically significant haemoglobinopathies using electrospray mass spectrometry-mass spectrometry

Increasing demand for population screening for the haemoglobinopathies gives rise to a requirement for high throughput systems, which allow for cost effective, rapid, sensitive and specific screening of clinically significant haemoglobins. We have developed a practical and efficient approach using tryptic digestion and electrospray triple quadrupole mass spectrometry-mass spectrometry (MSMS) in multiple reaction monitoring acquisition mode for the identification of the clinically important haemoglobin variants, S, C, DPunjab, OArab, and E. A total of 200 blood samples, comprising 52 haemoglobin AA, 57 AS (sickle cell trait), 44 AC (C trait), 16 SC (SC disease), 14 SS (sickle cell disease), 10 AE (E trait), 2 ADPunjab (DPunjab trait) and 1 each of AOArab (OArab trait), CC (C disease), DPunjabDPunjab (DPunjab disease), OArabOArab (OArab disease), and EE (E disease), have been analysed in parallel with existing phenotype and molecular methods. All haemoglobin variants were correctly identified by MSMS, with no false positives or false negatives. The system detects both heterozygotes and homozygotes and has potential applications in neonatal and antenatal screening.     

Self-care in Heart Failure Hospital Discharge Instructions—Differences Between Nurse Practitioner and Physician Providers

Patients with heart failure (HF) are at risk for frequent readmission potentially due to self-care deficits. Medical doctors (MDs) and nurse practitioners (NPs) both provide discharge instructions. However, each type of provider may emphasize different elements of care. The aim of this study was to analyze and compare the content of the documentation of 50 discharge instructions of heart failure patients written by NPs and MDs. Compared with MDs, NPs placed greater emphasis on symptom identification, and were more likely to advise and schedule follow-up appointments with primary care and cardiology providers rather than advising an appointment was needed without scheduling one.     

The development of microalbuminuria is associated with raised longitudinal adiponectin levels in female but not male adolescent patients with type 1 diabetes

AIMS/HYPOTHESIS: We determined the longitudinal relationship between adiponectin levels and the development of microalbuminuria in an inception cohort of children with type 1 diabetes. METHODS: Blood samples collected annually over a median of 9.0 (range 1.3-14.9) years were assayed for adiponectin and HbA(1c) in 55 children (36 girls) with type 1 diabetes and microalbuminuria whose age of onset of diabetes was 9.4 years (range 2.2-15.4). Samples were also assayed from normoalbuminuric children (controls) matched for age, sex and duration of diabetes. RESULTS: Overall, adiponectin levels were higher in girls than in boys, but only after 11 years of age (median [range]: 15.3 [5.8-124.4] vs 11.6 [4.1-26.5] mg/l, p < 0.001). Furthermore, adiponectin levels were higher in girls with microalbuminuria than in control girls, but this was only apparent after the onset of microalbuminuria (p = 0.001, adjusted for BMI, daily insulin dose, HbA(1c) and age). In boys, adiponectin levels did not differ between those with microalbuminuria and controls. Further sex-related discordant associations with adiponectin levels were observed; in girls, adiponectin levels were positively related to HbA(1c) levels (r = 0.2, p = 0.05) and urine albumin excretion (r = 0.3, p < 0.05) and inversely related to BMI (r = -0.2, p < 0.05). These associations were absent in boys. CONCLUSIONS/INTERPRETATION: In adolescent girls with type 1 diabetes but not in boys, adiponectin levels increase with increasing urine albumin excretion and onset of microalbuminuria. Although causal links cannot be inferred, this sexual dimorphism may reflect interactive effects of hyperglycaemia and sex steroids on risk of complications and adiponectin production.     

Pericardial influences on right and left ventricular filling dynamics

The influence of the pericardium on right and left ventricular filling was studied using two-dimensional and Doppler echocardiography in 14 open-chest dogs. Doppler echo parameters of filling included early (E) and late (A) velocities and their ratio (E/A) for the mitral and tricuspid valves. Right and left ventricular volumes were calculated from orthogonal two-dimensional echocardiographic images. Data were compared at three levels of left ventricular end-diastolic pressure (6 +/- 2, 13 +/- 3, and 21 +/- 4 mm Hg) at matched heart rates before and after pericardiectomy. The instantaneous diastolic pressure gradient was measured in 12 of the dogs. Pericardiectomy resulted in an increase in early mitral velocity, peak early diastolic pressure gradient, and E/A but not early mitral velocity normalized for end-diastolic volume. In contrast, for the tricuspid valve flow, pericardiectomy did not change E but caused a marked increase in A and a decrease in E/A. Right ventricular end-diastolic volumes at matched left ventricular end-diastolic volumes were similar before and after the pericardium was removed. However, removal of the pericardium caused a significant decrease of the slope for the right (86.0 +/- 27.0 x 10(-4) versus 50.0 +/- 19.5 x 10(-4) mm Hg/ml, p less than 0.01), but not left, ventricular ln end-diastolic pressure-volume relation (21.2 +/- 9.2 x 10(-3) versus 21.4 +/- 5.3 x 10(-3) mm Hg/ml, p = NS), and a decrease of the pressure intercept for the left (3.0 +/- 2.0 versus 1.6 +/- 0.9 mm Hg, p less than 0.05), but not right, ventricular ln end-diastolic pressure-volume relation (2.8 +/- 1.4 versus 1.4 +/- 0.8 mm Hg, p = NS). In conclusion, filling of the two ventricles is affected by the pericardium over a wide range of physiological ventricular volumes and pressures. At matched left ventricular end-diastolic volume, pericardiectomy causes a fundamental alteration in right, but not left, ventricular filling.     

Changes in platelet function with inflammation in patients undergoing vascular surgery

The role of platelets in ischaemic events is well established. Aspirin represents the default antiplatelet and blocks the metabolism of arachidonic acid (AA) at the cyclo-oxygenase enzyme (COX). AA is commonly used as a test of response to aspirin, but recent data raise uncertainty about the validity of this approach. Specifically, in some patients AA-induced clotting is not suppressed, but the level of COX-dependent AA metabolite, thromboxane B2 (TXB₂) is negligible. Furthermore, AA-induced whole blood clotting varies dynamically in individuals, who are aspirin responsive according to TXB₂ levels.

The aim of this study was to assess the level of AA-, ADP- and thrombin-mediated platelet reactivity in patients on aspirin before, during, and after major vascular surgery, which represents a model of on/off vascular inflammation. Firstly, we hypothesized, that in association with this inflammatory episode AA-, ADP- and thrombin-induced clotting would change in a dynamic manner. Secondly, that AA-induced clotting will be modified despite complete suppression of platelet TXB₂ production by aspirin throughout the periprocedural period, possibly via a lipoxygenase-mediated mechanism.

Fourty patients underwent major vascular surgery (open abdominal aortic aneurysm operation, infrainguinal bypass for subcritical limb ischaemia or peripheral aneurysm repair with bypass). They were all on 75 mg of aspirin prior to and throughout the perioperative period and received 5000 units of unfractionated heparin intraoperatively. AA-, ADP-, and thrombin-induced clotting, AA metabolites (TXB₂ and 12-Hyroxyeicosatetraenoic acid (12-HETE)) and inflammatory markers (CRP, IL-6, TNF-α and CD40) were measured pre-procedure and at 2, 24, 48 hours, 3 to 5 days and 3 months after surgery. AA-, ADP- and thrombin-induced platelet reactivity was assessed using thrombelastography. TXB₂, 12-HETE, IL-6, TNF-α, CD40 were determined using the sequential competitive binding Enzyme-Linked ImmunoAssay technique and CRP was determined using an immune-turbidimetric test on human serum.

There was a transient rise in inflammatory markers in the early perioperative period (CRP at 24, 48 hours and 3 to 5 days p < 0.001 and IL-6 at 2, 24, 48 hours and 3 to 5 days p < 0.001 as compared to baseline). Patients had negligible levels of TXB₂ throughout, confirming a consistent therapeutic response to aspirin. There was a transient rise in thrombin-mediated clotting (MA_Thrombin at 48 hours p = 0.001 and 3 to 5 days p < 0.001) and a fall in AA- and ADP-induced clotting in the early post op period (both MA_AA and MA_ADP p = 0.001 at 2 hours). At 3 months, the level of AA- and ADP-induced clotting was significantly higher than at baseline (p = 0.008 for MA_AA and p = 0.002 for MA_ADP), hence demonstrating a rebound effect.

These data demonstrate a novel dynamic variation in platelet aggregation with acute vascular inflammation, including AA-induced whole blood clotting which is apparently COX-1 independent.     

Association of body composition and lung function in children with cystic fibrosis

Survival in cystic fibrosis has improved significantly in the last 30 years, with major therapeutic goals of delaying the progressive loss of pulmonary function and maintaining normal growth. Dual-energy X-ray absorptiometry (DEXA) was performed in children with cystic fibrosis (CF) to assess both bone mineral density and body composition. We hypothesised that there would be an association between body composition and pulmonary function in children with CF. Fifty subjects with CF (28 males), mean age 12.7 years, participated in the study. Body composition was determined by DEXA. Body mass index (BMI) was calculated from the ratio of weight/height2 (kg/m2). Lung function was assessed by spirometry. Most patients (78%) had mild lung disease. The mean forced expired volume in 1 sec percent predicted (FEV1% predicted) for the 50 patients was 79.2% (range, 24-117%). There was a strong association between FEV1% predicted and BMI (R=0.59, P=0.0001). Fat-free mass had positive association with pulmonary function tests (R=0.30, P=0.03). Although fat mass showed a positive correlation with pulmonary function, this association did not reach statistical significance. In our group of children with CF and mild lung disease, pulmonary function was more strongly associated with BMI than with fat and fat-free mass.     

Motor unit properties from three synergistic muscles during ramp isometric elbow extensions

Many tasks require synergistic activation of muscles that possess different architectural, mechanical, and neural control properties. However, investigations of the motor unit (MU) mechanisms which modulate force are mostly restricted to individual muscles and low forces. To explore the pattern of MU recruitment and discharge behavior among three elbow extensors (lateral and long heads of the triceps brachii, and anconeus) during ramp isometric contractions, recruitment thresholds of 77 MUs in five young men were determined and corresponding MU discharge rates were tracked in 1-s epochs over forces ranging from 0 to 75 % of maximal voluntary isometric force (MVC). Across all forces, MUs in the lateral head discharged at higher rates than those in the anconeus (p < 0.001, Δ = 0.23). When all MUs were considered, recruitment thresholds in the long head of the triceps brachii were higher than the lateral head (p < 0.05, Δ = 0.70) with a trend (p = 0.08, Δ = 0.48) for higher recruitment thresholds in the long head compared with the anconeus. Together, these data indicate a potential mechanical disadvantage of the long head of the triceps brachii at 0° shoulder flexion. However, among low-threshold MUs (<10 % MVC), recruitment thresholds were lower in the anconeus than in both heads of the triceps brachii consistent with the expected twitch contractile and fiber type differences among these muscles. These findings illustrate the importance of considering synergistic relations among muscles used for a coordinated task, and the sensitivity of synergies to muscle architectural, mechanical, and possibly specific synaptic input factors.     

Regional Intraosseous Administration of Prophylactic Antibiotics is More Effective Than Systemic Administration in a Mouse Model of TKA

Background

In human TKA studies, intraosseous regional administration (IORA) of prophylactic antibiotics achieves local tissue antibiotic concentrations 10 times greater than systemic administration. However, it is unclear if such high concentrations provide more effective prophylaxis.

Questions/purposes

We asked: (1) What prophylaxis dosage and route (intravenous [IV] versus IORA of prophylactic antibiotics) produce less in vivo bacterial burden compared with no-antibiotic controls? (2) Compared with controls, what prophylaxis dosage and route yield fewer colony-forming units (CFUs) in euthanized animals in a model of TKA? (3) Is prophylactic IORA of antibiotics more effective than same-dose IV antibiotic administration in reducing CFUs?

Methods

Mice (six to nine per group) were block randomized to one of six prophylaxis regimens: control, systemic cefazolin (C100_IV), IORA of cefazolin (C100_IORA), systemic vancomycin (V110_IV), low-dose systemic vancomycin (V25_IV), and low-dose IORA of vancomycin (V25_IORA). Surgery involved placement of an intraarticular knee prosthesis, followed by an inoculum of bioluminescent Staphylococcus aureus strain Xen36. Biophotonic imaging assessed in vivo bacterial loads, and after 4 days bacterial load was quantified using culture-based techniques. Comparisons were made for each prophylactic regimen to controls and between same-dose IV and IORA of prophylactic antibiotic regimens.

Results

Mice treated with systemic high-dose vancomycin, IORA of vancomycin, or IORA of cefazolin had lower in vivo Staphylococcus aureus burdens (median area under curve, Control: 5.0 × 10⁶; V110_IV: 1.5 × 10⁶, difference of medians 3.5 × 10⁶, p = 0.003; V25_IV: 1.94 × 10⁶, difference 3.07 × 10⁶, p = 0.49; V25_IORA: 1.51 × 10⁶, difference 3.5 × 10⁶, p = 0.0011; C100_IORA: 1.55 × 10⁶, difference 3.46 × 10⁶, p = 0.0016; C100_IV: 2.35 × 10⁶, difference 2.66 × 10⁶, p = 0.23.) Similar findings were seen with culture-based techniques on recovered implants. IORA of prophylactic antibiotics was more effective than same-dose IV administration in reducing bacterial load on recovered implants (median CFUs < 7.0 × 10⁰ vs 2.83 × 10², p = 0.0183).

Conclusions

IORA of prophylactic cefazolin and vancomycin was more effective than the same dose of antibiotic given systemically. The effectiveness of vancomycin in particular was enhanced by IORA of prophylactic antibiotics despite using a lower dose.

Clinical relevance

Our study supports previous studies of IORA of prophylactic antibiotics in humans and suggests this novel form of administration has the potential to enhance the effectiveness of prophylaxis in TKA. Because of concerns regarding antibiotic stewardship, IORA of prophylactic vancomycin may be more appropriately restricted to patients having TKA who are at greater risk of infection, and clinical trials are in progress.