Are we meeting the needs of vulnerable children? Distribution of speech-language pathology services on the Gold Coast, Australia.

BACKGROUND: Compared to national averages, the Gold Coast, Australia, has a proportionately higher number of children entering their first year of primary school with 'at risk' or 'vulnerable' language skills. This paper investigates the distribution of paediatric speech-language pathology (SLP) services on the Gold Coast, relative to children's language and cognitive skills, and socioeconomic status (SES). METHODS: SLP service locations were identified through national association data and a manual search and mapped against SES and Australian Early Development Census data, for language and cognitive skills. RESULTS: Data for 7595 children was included, with 943 (12.4%) at risk and 780 (12.6%) vulnerable for language and cognitive skills. A total of 75 SLPs were identified (85.3% private, 14.6% public), which is 1 SLP to every 23 children who might have current or impending speech, language and communication needs. Fewer services were available in areas where vulnerable children were located and most were private providers, leading to further potential barriers to service access. CONCLUSIONS: The number of SLP services located on the Gold Coast is inadequate to meet the needs of children with language and cognitive skill vulnerabilities. Consideration of how services might be distributed is explored and warrants further consideration.

Management of children with chronic wet cough and protracted bacterial bronchitis: CHEST guideline and expert panel report

BACKGROUND: Wet or productive cough is common in children with chronic cough. We formulated recommendations based on systematic reviews related to the management of chronic wet cough in children (aged ≤ 14 years) based on two key questions: (1) how effective are antibiotics in improving the resolution of cough? If so, what antibiotic should be used and for how long? and (2) when should children be referred for further investigations? METHODS: We used the CHEST expert cough panel's protocol for systematic reviews and the American College of Chest Physicians (CHEST) methodologic guidelines and GRADE framework (the Grading of Recommendations Assessment, Development and Evaluation). Data from the systematic reviews in conjunction with patients' values and preferences and the clinical context were used to form recommendations. Delphi methodology was used to obtain consensus for the recommendations/suggestions made. RESULTS: Combining data from the systematic reviews, we found high-quality evidence in children aged ≤ 14 years with chronic (> 4 weeks' duration) wet/productive cough that using appropriate antibiotics improves cough resolution, and further investigations (eg, flexible bronchoscopy, chest CT scans, immunity tests) should be undertaken when specific cough pointers (eg, digital clubbing) are present. When the wet cough does not improve following 4 weeks of antibiotic treatment, there is moderate-quality evidence that further investigations should be considered to look for an underlying disease. New recommendations include the recognition of the clinical diagnostic entity of protracted bacterial bronchitis. CONCLUSIONS: Compared with the 2006 Cough Guidelines, there is now high-quality evidence for some, but not all, aspects of the management of chronic wet cough in specialist settings. However, further studies particularly in primary health) are required.

Variation in the circularly polarized light reflection of Lomaptera (Scarabaeidae) beetles.

An extended spectroscopic study on the left-through-left circularly polarized reflection spectra of a large number of beetles from the Australasian Scrabaeidae:Cetoniinae of the Lomaptera genus was undertaken. We have obtained a five-category spectral classification. The principal spectral features, which even within the genus range from blue to infrared, are related to structural chirality in the beetle shells. The detailed features of each spectral classification are related to different structural perturbations of the helix, including various pitch values and abrupt twist defects. These spectral characteristics and associated shell structures are confirmed on the basis of simple modelling. An important conclusion from our study is that the simple helical structure resulting in a single symmetric Bragg peak is not the dominant spectral type. Rather the reality is a rich tapestry of spectral types. One intriguing specimen is identified via a scanning electron micrograph to consist of a double interstitial helix leading to a particular double-peak spectrum.

New IR imaging modalities for cancer detection and for intra-cell chemical mapping with a sub-diffraction mid-IR s-SNOM.

We present two new modalities for generating chemical maps. Both are mid-IR based and aimed at the biomedical community, but they differ substantially in their technological readiness. The first, so-called "Digistain", is a technologically mature "locked down" way of acquiring diffraction-limited chemical images of human cancer biopsy tissue. Although it is less flexible than conventional methods of acquiring IR images, this is an intentional, and key, design feature. It allows it to be used, on a routine basis, by clinical personnel themselves. It is in the process of a full clinical evaluation and the philosophy behind the approach is discussed. The second modality is a very new, probe-based "s-SNOM", which we are developing in conjunction with a new family of tunable "Quantum Cascade Laser" (QCL) diode lasers. Although in its infancy, this instrument can already deliver ultra-detailed chemical images whose spatial resolutions beat the normal diffraction limit by a factor of ∼1000. This is easily enough to generate chemical maps of the insides of single cells for the first time, and a range of new possible scientific applications are explored.

Associations between hoof shape and the position of the frontal plane ground reaction force vector in walking horses.

AIMS: To determine the frontal plane position of the ground reaction force vector at its centre of pressure under the hoof of walking horses, and its projection through the distal limb joints, and to relate this to hoof geometric measurements. METHODS: Reflective markers were glued to the forelimb hooves and skin of 26 horses, over palpable landmarks representing centres of the coffin, fetlock and carpal joints, and the dorsal toe at its most distal point. A 4-camera kinematic system recorded the position of these markers as the horse walked in hand across a force platform, to generate a frontal plane representation of the ground reaction force vector passing between the markers at the joints. The position of the vector was calculated as the relative distance between the lateral (0%) and medial (100%) markers at each joint. Digital photos were taken of the hoof in frontal and sagittal views to determine hoof geometric measurements. Associations between these and the position of the force vector at each joint were examined using Pearson correlation coefficients. RESULTS: Mean vector position for both forelimbs at the toe, coffin, fetlock and carpal joint was 50.1 (SD 8.9), 53.0 (SD 9.2), 54.6 (SD 11.4) and 50.5 (SD17.3)%, respectively, of the distance between the lateral and medial sides of the joint in the frontal plane. Across all four joints, the vector position was slightly more medial (2-4%) for the right than left limb (p>0.05). Medial hoof wall angle was correlated (p<0.05) with force vector position at the fetlock (r=-0.402) and carpal (r=-0.317) joints; lateral hoof wall angle with vector position at the toe (r=0.288) and carpal (r=-0.34) joint, and medial hoof wall height with vector position at the fetlock (r=-0.306) and carpal (r=-0.303) joints. CONCLUSION: The position of the two-dimensional frontal plane ground reaction force vector at the toe, and at the fetlock and carpal joints was associated with hoof shape. Mediolateral hoof balance has been shown in vitro to affect articular forces, which may be a factor in development of joint disease. The effect of hoof shape needs to be evaluated at faster gaits to determine the potential for joint injury in the presence of larger forces.

Seasonal and annual influence on insulin and cortisol results from overnight dexamethasone suppression tests in normal ponies and ponies predisposed to laminitis.

REASONS FOR PERFORMING STUDY: A simple, accurate test for identifying individual animals at increased risk of laminitis would aid prevention. Laminitis-prone ponies have a greater serum insulin response to dexamethasone administration than normal ponies in the summer, but the response during different seasons is unknown. OBJECTIVE: To test the hypothesis that previously laminitic ponies have a greater insulin response to dexamethasone than normal ponies, which is present during all seasons. STUDY DESIGN: Prospective longitudinal study. METHODS: Overnight dexamethasone suppression tests were performed on 7 normal ponies and 5 previously laminitic ponies in spring 2009 and 2010, summer 2008 and 2010, autumn 2009 and winter 2008, while the ponies were at pasture. In spring 2010, a dexamethasone suppression test was performed after the ponies had been fed only hay for 3 weeks. Serum cortisol and insulin concentrations pre- and post dexamethasone were measured. Linear mixed models were used to analyse the data. RESULTS: Insulin concentrations pre- and post dexamethasone were significantly higher in previously laminitic ponies than in normal ponies during spring 2009 and summer 2008, but there was no difference between groups in spring 2010, summer 2010, autumn 2009 or winter 2008. Insulin concentration varied significantly with season. Diet had no apparent effect on insulin concentration pre- or post dexamethasone in spring 2010. Cortisol concentrations post dexamethasone were significantly higher in previously laminitic ponies than in normal ponies in autumn 2009, with concentrations above the reference range (<25 nmol/l) in both groups in summer 2008 and autumn 2009. Individual ponies had insufficient cortisol suppression in all seasons. CONCLUSIONS: There were significant differences between groups in insulin and cortisol concentrations post dexamethasone during some seasons, but this was not present in all years. Wide interindividual variation in response limits the usefulness of a dexamethasone suppression test for predicting the susceptibility of an individual animal to laminitis. POTENTIAL RELEVANCE: Abnormal insulin and cortisol responses to dexamethasone must be interpreted in the light of the individual animal, seasonal and annual variation reported here.

Seasonal variation in maintenance of phenylephrine-induced tone in isolated equine digital arteries under hypoxic or hyperoxic conditions in vitro.

Digital vasoconstriction, ischaemia and hypoxia may predispose to acute laminitis. Laminitis incidence varies seasonally, peaking in spring and summer. Direct seasonal influences on equine digital artery (EDA) contractility have not been investigated. This study assessed seasonal variation in maintenance of phenylephrine (PHE)-induced tone in isolated EDAs under hypoxic (95% nitrogen) and hyperoxic (95% oxygen) conditions. The objective was to measure change in arterial tone over time after constriction to a plateau with PHE. Tone was measured at plateau and over time and percentage change calculated. Hyperoxic EDAs maintained PHE-induced tone over 1 h with no seasonal variation. Hypoxic EDAs relaxed in fall (median [inter-quartile range] 59% [44-77%] decrease from plateau; P=0.008), contracted in spring (65% [20-192%] increase from plateau; P=0.03) and did not significantly change tone in winter (18% [0-28%] decrease; P=0.13). Continued contraction under hypoxic conditions in spring may contribute to digital vasoconstriction.

Clinical signs and symptoms of oropharyngeal aspiration and dysphagia in children.

The diagnostic value of various signs and symptoms (clinical markers) in predicting oropharyngeal aspiration (OPA) or swallowing dysfunction has not been established in children. The present retrospective study was undertaken to: 1) identify specific clinical markers associated with radiographic evidence of OPA, isolated laryngeal penetration (ILP) and post-swallow residue (PSR); 2) determine the sensitivity and specificity of clinical markers associated with OPA; and 3) determine the influence of age and neurological impairment on clinical markers of OPA. In total, 11 clinical markers of dysphagia were compared with the videofluoroscopic swallow study (VFSS) results (OPA, ILP and PSR) in 150 children on diets of thin fluid and purée consistencies. Chi-squared and logistic regression were used to analyse the association between clinical markers and VFSS-identified swallowing dysfunction. In children with OPA, wet voice (odds ratio (OR) 8.90, 95% confidence interval (CI) 2.87-27.62), wet breathing (OR 3.35, 95% CI 1.09-10.28) and cough (OR 3.30, 95% CI 1.17-9.27) were significantly associated with thin fluid OPA. Predictive values included: wet voice (sensitivity 0.67; specificity 0.92); wet breathing (sensitivity 0.33; specificity 0.83); and cough (sensitivity 0.67; specificity 0.53). No clinical markers were significantly associated with OPA, ILP or PSR on the purée consistency. Cough was significantly associated with PSR on thin fluids (OR 3.59, 95% CI 1.22-10.55). Differences were found for age. Wet voice, wet breathing and cough were good clinical markers for children with oropharyngeal aspiration on thin fluid but not on purée. Age and neurological status influenced the significance of these clinical markers.

The effects of acute whole body vibration as a recovery modality following high-intensity interval training in well-trained, middle-aged runners.

The main purpose of the present study was to examine the effects of acute whole body vibration (WBV) on recovery following a 3 km time trial (3 km TT) and high-intensity interval training (HIIT) (8 x 400 m). Post-HIIT measures included 3 km time-trial performance, exercise metabolism and markers of muscle damage (creatine kinase, CK) and inflammation (c-reactive protein, CRP). A second purpose was to determine the effects of a 3 km TT and HIIT on performance and metabolism the following day. Nine well-trained, middle-aged, male runners [(mean +/- SD) age 45 +/- 6 years, body mass 75 +/- 7 kg, VO2peak 58 +/- 5 ml kg(-1 )min(-1)] performed a constant pace run at 60 and 80% velocity at VO2peak (v VO2peak) followed by a 3-km TT and a 8 x 400-m HIIT session on two occasions. Following one occasion, the athletes performed 2 x 15 min of low frequency (12 Hz) WBV, whilst the other occasion was a non-WBV control. Twenty-four hours after each HIIT session (day 2) participants performed the constant pace run (60 and 80% v VO2peak) and 3 km TT again. There was a significant decrease in 3 km TT performance (~10 s) 24 h after the HIIT session (P < 0.05); however, there were no differences between conditions (control vs. vibration, P > 0.05). Creatine kinase was significantly elevated on day 2, though there were no differences between conditions (P > 0.05). VO2peak and blood lactate were lower on day 2 (P < 0.05), again with no differences between conditions (P > 0.05). These results show no benefit of WBV on running performance recovery following a HIIT session. However, we have shown that there may be acute alterations in metabolism 24 h following such a running session in well-trained, middle-aged runners.

Restriction of oral intake of water for aspiration lung disease in children.

BACKGROUND: Primary aspiration of food and fluid is commonly seen in children with feeding and swallowing difficulties associated with a range of diseases and complex medical conditions. Respiratory sequelae and pneumonia are known to be associated with primary aspiration of ingested material, however causality between primary aspiration of specific food and fluid types and pulmonary effects in children is yet to be established in controlled trials. The relative pulmonary morbidity of aspiration of ingested food and fluid materials versus other causes of respiratory disease such as viral and bacterial causes, secondary aspiration of gastrointestinal contents and predisposing lung conditions such as chronic neonatal lung disease in a developing immune system is also unclear. Current management decisions for children who aspirate have to optimise oral nutrition and hydration, while reducing the risk of aspiration to preserve pulmonary integrity. This generally includes restricting aspirated food or fluids and providing texture-modified diets and thickened fluids. Young children frequently refuse thickened fluids providing a management dilemma for both families and health professionals. OBJECTIVES: Our objective was to evaluate the efficacy of restriction of oral water ingestion on the pulmonary status of children with thin fluid aspiration demonstrated on a modified barium swallow study. SEARCH STRATEGY: The Cochrane Central Register of Controlled Trials (CENTRAL), the Cochrane Airways Collaborative Review Group Specialised Register, MEDLINE, EMBASE and CINAHL databases were searched by the Cochrane Airways Group. The latest search was performed in January 2005. SELECTION CRITERIA: All randomised controlled trials comparing restriction of oral intake of water with unlimited oral water ingestion were eligible to be included. DATA COLLECTION AND ANALYSIS: Results of searches were reviewed against a pre-determined criteria for inclusion. No eligible trials were identified for a paediatric population and thus no data were available for analysis. One trial in an adult population was identified and reported. MAIN RESULTS: No randomised controlled trials examining the efficacy of restriction of oral intake of water in the management of children with thin fluid aspiration were found. In a single study in an adult population with stroke, no significant differences were seen between a control group of oral water restriction and the experimental group of unlimited oral water ingestion on outcomes such as pneumonia, total oral fluid intake and dehydration. AUTHORS' CONCLUSIONS: There are no trials that have adequately evaluated the pulmonary effects of allowing or restricting oral water ingestion in children known to have primary aspiration of thin fluids. Thus, there is currently an absence of evidence to support a strict approach of full restriction of oral intake of water or support a more liberal approach of allowing oral water ingestion in children with primary aspiration of thin fluids.

Enzymatic bioremediation: from enzyme discovery to applications.

1. Enzymatic bioremediation is potentially a rapid method of removing environmental pesticide residues. Applications include the treatment of residues resulting from agricultural production and processing industries, such as the treatment of irrigation waters, surface-contaminated fruit and vegetables and spent dip liquors. 2. A specific application for some organophosphate-degrading enzymes involves detoxification of nerve agent stockpiles. Effective and affordable remediation requires highly specialized enzymes, so protein engineering techniques are being used to improve properties of various source enzymes to enhance catalytic rates, stability and substrate range. 3. Trials with an optimized organophosphate-degrading enzyme have shown the feasibility of such technology in various applications. 4. The enzymes developed for environmental remediation for specific pesticide classes also have applications as antidotes for high-dose pesticide poisonings and as prophylaxis for people at risk of high pesticide doses.

Number of viable CD34(+) cells reinfused predicts engraftment in autologous hematopoietic stem cell transplantation.

Reduced CD34(+) cell viability due to cryopreservation has unknown effects on subsequent hematopoietic engraftment in autologous transplantation. Thirty-six consecutive autologous peripheral stem cell collections were analyzed for absolute viable CD34(+) cell numbers at the time of stem cell collection and prior to re-infusion. Viable CD34(+) cells were enumerated using single platform flow cytometry and the molecular exclusion dye 7-amino actinomycin D. The median number of viable CD34(+) cells was 3.6 x 10(6)/kg at the time of harvest and 2.0 x 10(6)/kg after thawing. When viable CD34(+)cells enumerated after thawing were <2.0, 2.0-5.0, or >5.0 x 10(6)/kg, the median time to platelet engraftment was 17, 12 and 10 days, respectively (P < 0.05 for comparison of the group with <2.0 x 10(6)/kg and the other two groups), and the median time to neutrophil engraftment was 13, 14 and 12 days, respectively (P = NS). A minimum of 2.0 x 10(6) CD34(+) cells/kg was harvested in 33 of 36 patients (92%) but only 19 of 36 (52%) patients met this threshold at the time of reinfusion. The reduced numbers of viable CD34(+) cells measured prior to re-infusion is associated with time to platelet engraftment and may be useful in monitoring stem cell loss during processing and identifying patients at risk of graft failure.

Enrichment of a microbial culture capable of degrading endosulphate, the toxic metabolite of endosulfan.

AIMS: The aim of this study was to isolate a source of enzymes capable of degrading endosulphate (endosulfan sulphate), the toxic metabolite of the pesticide endosulfan. METHODS AND RESULTS: A microbial broth culture capable of degrading endosulphate was enriched from endosulfan-contaminated soil by providing the metabolite as the sole source of sulphur in broth culture. No microbial growth was observed in the absence of endosulphate. In the presence of endosulphate, growth of the culture occurred with the concomitant formation of three chlorine-containing compounds. Thin layer chromatography and gas chromatography--mass spectral analysis identified these metabolites as endosulfan monoaldehyde, 1,2,3,4,7,7-hexachloro-5,6-bis(methylene)bicyclo[2.2.1]-2-heptene and 1,2,3,4,7,7-hexachloro-5-hydroxymethylene-6-methylenebicyclo[2.2.1]-2-heptene. The second and third compounds have not been reported in previous metabolic studies. The enriched culture was also able to utilize alpha- and beta-endosulfan as sulphur sources, each producing the hydrolysis product endosulfan monoaldehyde as the sole chlorine-containing metabolite. Alpha-endosulfan was more readily hydrolysed than the beta-isomer. CONCLUSIONS: This study isolated a mixed microbial culture capable of degrading endosulphate. The products of degradation were characterized as novel endosulfan metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the isolation of a mixed microbial culture that is potentially a valuable source of hydrolysing enzymes for use in enzymatic bioremediation, particularly of endosulphate and alpha-endosulfan residues.

UV-induced changes in the skin: can they be repaired?

The damaging effects of UVB light have been described previously and include a number of changes to multiple cell types. At previous meetings of this society, we have shown that Langerhans' cells are the most susceptible to UVB induced damage which can be shown as ultrastructural changes in dendrites, nucleus and cytoplasm by transmission electron microscopy. We have also shown that their patterns of migration from skin to regional lymph node and their ability to present antigens to autologous T cells have been profoundly altered by UVB irradiation. The aim of this work was to establish if it was possible to reverse any of the damage done to Langerhans' cells by UVB exposure by topical application of a DNA repair enzyme such as T4N5 endonuclease. These experiments were undertaken in a sheep model that allowed collection of cells as they migrate from the skin. This allowed for a direct examination of the migration characteristics and ultrastructural features of all Langerhans' cells before, during, and for 2 weeks after exposure to a single dose of UVB. Results obtained from this project indicate that treatment by topical application of DNA repair enzyme immediately after UVB irradiation may restore a number of normal immune parameters associated with the structure and function of migrating Langerhans' cells. It appears that there is a dose related correction of the increased tempo of cell migration and some improvements in the number of ultrastructurally damaged Langerhans' cells have also been associated with application of higher doses of DNA repair enzyme. These preliminary findings indicate that some potential therapeutic benefits are associated with the use of such agents in reversing the immunological damage caused by exposure to erythemal doses of UVB light.

Nitric oxide deficiency in fenfluramine- and dexfenfluramine-induced pulmonary hypertension.

Dexfenfluramine and fenfluramine greatly increase the risk of developing pulmonary hypertension (PHT). The mechanism of anorexigen-associated PHT (AA-PHT) and the reason PHT occurs in a minority of people exposed are unknown. Anorexigens are weak pulmonary vasoconstrictors, but they become potent when synthesis of the endogenous vasodilator nitric oxide (NO) is suppressed. We hypothesized NO deficiency predisposes affected individuals to develop AA-PHT. A prospective, case-control, study was performed on consecutive patients with AA-PHT (n = 9). Two sex-matched control groups were selected: patients with primary PHT (P-PHT, n = 8) and normal volunteers (n = 12). Lung NO production (VNO) and systemic plasma oxidation products of NO (NOx) were measured at rest and during exercise. AA-PHT developed 17 +/- 6 mo after a short course of anorexigen (6 +/- 2 mo) and was irreversible. VNO was lower in AA-PHT than in P-PHT and correlated inversely with PVR (p < 0.05). The apparent VNO deficiency may have resulted from increased oxidative inactivation of NO in patients with AA-PHT, as their NOx levels were elevated (p < 0.05) in inverse proportion to VNO (r2 = 0. 55; p < 0.02). In susceptible persons, anorexigens can cause an irreversible syndrome of PHT, hypoxemia, and systemic vascular complications after brief exposures. These patients have a relative NO deficiency years after discontinuing the anorexigen, perhaps explaining their original susceptibility.

Body weight does not predict for anti-Xa levels after fixed dose prophylaxis with enoxaparin after orthopedic surgery.

Enoxaparin after joint arthroplasty is effective prophylaxis against venous thromboembolism. This is usually given as a fixed dose without monitoring of anti-Xa levels. This study assesses the relationship between trough anti-Xa levels, body weight, and venous thromboembolism. Consenting patients at three institutions were treated with Enoxaparin 30 mg subcutaneously bis in die postoperatively until discharge. Chromogenic anti-Xa levels were measured on the fifth postoperative day by the method of Stachrome (Diagnostica Stago). All patients had bilateral compression doppler ultrasonography on day 10 or discharge and were followed for 12 weeks for evidence of venous thromboembolism. Eleven patients developed objectively confirmed venous thromboembolism during the study. In this study, there was poor correlation between weight and anti-Xa levels. In addition, body weight and anti-Xa levels of patients who developed venous thromboembolism were compared to those who did not and there were no significant differences between the two groups. In conclusion, this study shows that there is poor correlation of trough anti-Xa levels with body weight. Recognizing the low overall event rate this study does not support the need to monitor anti-Xa levels or adjusting the dose according to weight.

Silicone gel enhances the development of autoimmune disease in New Zealand black mice but fails to induce it in BALB/cAnPt mice.

Anecdotal evidence links silicone gel breast implants with the development of autoimmune connective tissue disease in women. To investigate whether silicone gel is capable of directly inducing and/or enhancing the development of autoimmune disease, female BALB/cAnPt (BALB/c) and New Zealand Black (NZB) mice were injected subcutaneously with silicone gel, pristane, a nonmetabolizable substance that can cause plasmacytomas in BALB/c and NZB mice, or saline and monitored for the development of glomerulonephritis and autoantibody production. NZB, but not BALB/c, mice spontaneously develop autoantibodies and an autoimmune hemolytic anemia by 12 months of age. Over a period of 10 months, biweekly screening for proteinuria revealed increases in urinary protein in NZB mice that received multiple injections of either silicone gel or pristane. In contrast, urinary protein was unaffected in identically treated BALB/c mice. Although, silicone gel had no effect on serum titers of antierythrocyte antibodies in NZB mice, the hematocrits were significantly decreased. Moreover, silicone gel both increased the concentration of IgM anti-type I collagen antibodies and skewed the immunofluorescent staining pattern of serum autoantibodies on HEp-2 cells. In contrast, silicone gel failed to induce the production of anti-erythrocyte or antinuclear antibodies in BALB/c mice and induced only slight increases in IgG anti-type I collagen antibodies. These results suggest that silicone gel can exacerbate the development of autoimmune disease in autoimmune NZB mice, but fails to induce disease in normal BALB/c mice. This is consistent with several epidemiological studies failing to demonstrate an increase in the incidence of autoimmune disease in women with breast implants. However, because silicone gel was able to exacerbate autoimmune disease in NZB mice, it may play a similar role in the development of autoimmune disease in a small percentage of women who are genetically susceptible to such diseases.

Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines. International Society of Hematotherapy and Graft Engineering.

In concert with the International Society of Hematotherapy and Graft Engineering (ISHAGE), we previously described a set of guidelines for detection of CD34+ cells based on a four-parameter flow cytometry method (CD45 FITC/CD34 PE staining, side and forward angle light scatter). With this procedure, an absolute CD34+ count is generated by incorporating the leukocyte count from an automated hematology analyser (two-platform method). In the present study, we modified the basic ISHAGE method with the addition of a known number of Flow-Count fluorospheres. To reduce errors inherent to sample washing/centrifugation, we implemented ammonium chloride lyse, no-wash no-fix sample processing. These modifications convert the basic protocol into a single-platform method to determine the absolute CD34 count directly from a flow cytometer and form the basis of the Stem-Kit from Coulter/Immunotech. A total of 72 samples of peripheral blood, apheresis packs, and cord blood were analysed and compared using the ISHAGE protocol with or without the addition of fluorescent microspheres. Comparison of methods showed a high correlation coefficient (r=0.99), with no statistically significant difference or bias between methods (P > 0.05). Linearity of the absolute counting method generated an R2 value of 1.00 over the range of 0-250/microl. Precision of the absolute counting method measured at three concentrations of CD34+-stabilised KG1 a cells (Stem-Trol, COULTER) generated a coefficient of variation (C.V.) ranging from 4% to 9.9%. In a further modification of the single-platform method, the viability dye 7-amino actinomycin D was included and demonstrated that both viable and nonviable CD34+ cells could be identified and quantitated. Together, these modifications combine the accuracy and sensitivity of the original ISHAGE method with the ability to produce an absolute count of viable CD34+ cells. It is the accurate determination of this value that is most clinically relevant in the transplant setting. These modifications may improve the interlaboratory reproducibility of CD34 determinations due to the reduction in sample handling and calculation of results.