Ambulatory blood pressure monitoring in pheochromocytoma - paraganglioma: A single center experience.

Context/Aims: Pheochromocytoma and paraganglioma (PPGL) are rare tumors, and data on ambulatory blood pressure monitoring (ABPM) in these patients and the effect of blocking on ABPM parameters is limited. We aimed to describe ABPM parameters in a cohort of PPGL at our center in western India. Methods: Retrospective study of patients with PPGL whose ABPM data was available. Demographic details, secretory status, and ABPM data were retrieved. Coefficient of variability (CV) was calculated as standard deviation/mean in percentage. Results: In the 39 included patients, mean age at presentation was 39.3 ± 14.2 yr; 20 (51.3%) were males, 25 (64.1%) hypertensive, and mean tumor diameter was 5.3 cm. In 18 patients whose baseline ABPM was done without medications, those with nocturnal blood pressure dipping (6/18, 33%) had higher serum metanephrines (median 313.2 vs. 34.7 pg/ml, P = 0.028). Despite normal office blood pressure (BP), 8.9% of systolic BP readings were >140 mmHg, and 1.2% were >160 mmHg. Among 29 patients with both pre and post-block ABPM, mean BP (systolic 121.6 vs. 132.5 mmHg, P = 0.014; diastolic 68.9 vs. 76.4 mmHg, P = 0.005) and percentage of BP readings above 140 mmHg (median 9.4% vs. 24.4%, P = 0.016) were significantly lowered after the preoperative blockade in hypertensive (n = 19) patients, whereas CV was similar. The post-blockade ABPM characteristics were similar in patients blocked with amlodipine or prazosin. Conclusion: ABPM provides additional information about BP characteristics in PPGL. The preoperative blocking decreases the magnitude of BP excursions but does not affect BP variability.

Thoracoscopic oesophageal atresia/tracheo-oesophageal fistula (OA/TOF) repair is associated with a higher stricture rate: a single institution's experience.

PURPOSE: Thoracoscopic OA/TOF repair was first described in 1999. Currently, less than 10% of surgeons routinely employ minimally access surgery. Our primary aim was to review our immediate-, early- and long-term outcomes with this technique compared with the open approach. METHODS: A retrospective review of all patients undergoing primary OA/TOF (Type C) repair at our institution from 2009 was conducted. Outcome measures included length of surgery, conversion rate from thoracoscopy, early complications such as anastomotic leak and post-operative complications such as anastomotic strictures needing dilatations. Fisher's exact and Kruskal-Wallis tests were used for statistical analysis. RESULTS: 95 patients in total underwent OA/TOF repair during the study period of which 61 (64%) were completed via an open approach. 34 were attempted thoracoscopically of which 11 (33%) were converted. There was only one clinically significant anastomotic leak in our series that took place in the thoracoscopic group. We identified a significantly higher stricture rate in our thoracoscopic cohort (72%) versus open surgery (43%, P < 0.05). However, the median number of dilations (3) performed was not significantly different between the groups. There was one recurrent fistula in the thoracoscopic converted to open group. Our median follow-up was 60 months across the groups. CONCLUSION: In our experience, the clinically significant leak rate for both open and thoracoscopic repair as well as recurrent fistula is much lower than has been reported in the literature. We do not routinely perform contrast studies and are, thus, reporting clinically significant leaks only. The use of post-operative neck flexion, ventilation and paralysis is likely to be protective towards a leak. Thoracoscopic OA/TOF repair is associated with a higher stricture rate compared with open surgery; however, these strictures respond to a similar number of dilatations and are no more refractory. Larger, multicentre studies may be useful to investigate these finding further.

The multi-disciplinary management of complex congenital and acquired tracheo-oesophageal fistulae.

AIM OF THE STUDY: Complex tracheo-oesophageal fistulae (TOF) are rare congenital or acquired conditions in children. We discuss here a multidisciplinary (MDT) approach adopted over the past 5 years. METHODS: We retrospectively collected data on all patients with recurrent or acquired TOF managed at a single institution. All cases were investigated with neck and thorax CT scan. Other investigations included flexible bronchoscopy and bronchogram (B&B), microlaryngobronchoscopy (MLB) and oesophagoscopy. All cases were subsequently discussed in an MDT meeting on an emergent basis if necessary. MAIN RESULTS: 14 patients were referred during this study period of which half had a congenital aetiology and the other half were acquired. The latter included button battery ingestions (5/7) and iatrogenic injuries during oesophageal atresia (OA) repair. Surgical repair was performed on cardiac bypass in 3/7 cases of recurrent congenital fistulae and all cases of acquired fistulae. Post-operatively, 9/14 (64%) patients suffered complications including anastomotic leak (1), bilateral vocal cord paresis (1), further recurrence (1), and mortality (1). Ten patients continue to receive surgical input encompassing tracheal/oesophageal stents and dilatations. CONCLUSIONS: MDT approach to complex cases is becoming increasingly common across all specialties and is important in making decisions in these difficult cases. The benefits include shared experience of rare cases and full access to multidisciplinary expertise.

Management of Paediatric Testicular Torsion - Are we adhering to Royal College of Surgeons (RCS) recommendations.

Introduction In 2015, the Royal College of Surgeons of England (RCS) commissioned the East Midlands Clinical Network to develop a set of guidelines for the management of paediatric torsion. Two quality measures identified were the provision of surgery locally where possible and 100% of explorations within three hours. We sought to assess the adherence to these quality measures within our referral network. Materials and methods Retrospective data were collected for all paediatric scrotal explorations performed within our centre between January 2014 and July 2016. Patient demographics, sources of referral, transfer times, time to surgery and operative findings were obtained. Results A total of 100 patients underwent a scrotal exploration. Median age at presentation was 11 years (range 4 months to 15 years). Fifty-three per cent of referrals were from network hospitals. The median duration of symptoms was 25 hours (range 1-210 hours). The median transfer time from local centres was 120 minutes (range 45-540 minutes). The median time to theatre from the decision being made to operate was 60 minutes (range 30-600 minutes). Eighty-seven per cent of cases were explored within three hours. There were 13 cases of torsion with one orchidectomy. When taking into account the transfer time for external patients aged over five years without precluding comorbidities, exploration within three hours dropped to 18 of 46 (39%). Conclusion The RCS guidelines recognise the need for specialist input in very young patients. A large proportion of explorations are, however, currently taking place in older patients with unacceptably long transfer times. We propose an extension of this review nationally to work towards the local provision of care for suitable patients.

Ultrasonographic Features of Ulnar Nerve Affected by Hansen's Disease.

Leprosy continues to be a major public health problem in some areas of our country. It predominantly afflicts peripheral nerves and skin and may lead to deformities. Social stigma as a result of deformities further plagues the situation. Prompt and early diagnosis coupled with adequate treatment, concurrent rehabilitative strategies if deformities do occur, and health education help to control the problem. Definitive diagnosis of leprosy has traditionally been based on assessment of slit skin smears (SSS) after AFB-staining and characteristic histopathology after biopsyof the lesion. However, recently, thickening of the peripheral nerves has been demonstrated by ultrasonography and this can be used as a sensitive tool to assess and measure enlargement of peripheral nerves, which are hallmarks for leprosy especially in clinical settings. In this report, the ultrasonographic findings of ulnar nerve enlargement due to leprosy in a fourteen-year-old male patient are described.

Post-natal outcomes of antenatally diagnosed intra-abdominal cysts: a 22-year single-institution series.

PURPOSE: The aim of this study was to establish the post-natal diagnosis and outcome of antenatally diagnosed intra-abdominal cysts between 1991 and 2013 at our institution. METHODS: All antenatally diagnosed intra-abdominal cysts between 1991 and 2013 were identified using a foetal anomaly database. The cysts were monitored for resolution. In all cases where the cyst had not resolved antenatally, additional post-natal scans were conducted. Antenatal diagnosis, post-natal diagnosis and outcomes were also recorded. RESULTS: 118 cases of antenatal intra-abdominal cysts were identified over the 22-year study period with a 98 % live birth rate. The overall accuracy of an antenatal diagnosis at our institution was 92 %. 26 cases (22 %) resolved spontaneously in utero, the majority of which (77 %) were ovarian in nature. Four tumour cases were identified in the series, which included two neuroblastomas, one yolk sac tumour and one teratoma. 90 cysts persisted post-natally with 52 % requiring surgery. These primarily included choledochal and enteric duplication cysts as well as symptomatic solid organ cysts. Diagnostic revision was limited to 8 % of cases over the study period with an overall improvement over the last decade. Overall, 40 % of all antenatally diagnosed cysts required surgical intervention. In those cysts that persisted post-natally, 52 % required surgery. CONCLUSIONS: A fifth of prenatally diagnosed intra-abdominal cysts will resolve with most ovarian cysts regressing in utero. Half of all persistent cysts will, however, require surgical intervention. These data are useful for prenatal counselling and demonstrates the important role played by the paediatric surgeon in the overall management of intra-abdominal cysts.

Circulating nucleic acids in the assessment of endogenous growth hormone production.

There is growing concern about the use of recombinant human growth hormone (rhGH) by individuals taking part in competitive sports. Although rhGH is banned by the international organizations, the detection of GH doping is difficult. We postulated that rhGH will suppress endogenous GH production, which can be assessed by the measurement of mRNA for GH and growth hormone-releasing hormone (GHRH). In order to prove this concept, we undertook a pilot study to examine whether circulating nucleic acids are useful in the detection of endogenous GH production. Blood samples were collected into PAXgene tubes from 37 healthy controls and 12 acromegalic patients. RNA was extracted from the samples, cDNA was obtained, and the quantities of mRNA for GH and GHRH were measured using real-time PCR. In acromegalic patients, median mRNA concentration for GHRH (corrected for beta-actin mRNA) was 30.7 times lower than in controls (median delta C(T)) value of -0.128 versus 3.927, P < 0.001). There was a significant correlation between serum IGF-1 SD score and mRNA for GHRH (r= 0.407). In acromegalic patients, mRNA for GH was significantly higher than in controls (median values of -4.694 versus -0.044, P < 0.05). As GH production is known to decline with age, we also examined mRNA for GH and GHRH according to age subgroups. Both markers were significantly lower in the older age group (>50 years) compared to the younger age group (<34 years). These results show that mRNA for GH and GHRH can be detected in the peripheral circulation and raises the possibility of using these markers in the detection of exogenously administered GH.

Stability of beta-actin mRNA in plasma.

It has long been thought that mRNA is labile and easily prone to degradation. However, a recent study demonstrated that GAPDH mRNA in cell-free plasma may remain stable up to 24 hours after blood collection. As there are no other independent studies, we attempted to reproduce the findings of that study. In our study, blood was collected from a healthy male volunteer into Vacutainer tubes containing EDTA. Blood samples were placed on ice and plasma separated by double-centrifugation at times 0, 1, 2, and 5 hours after blood collection. mRNA was extracted from four aliquots of the blood sample by means of the QIAamp Viral RNA kit. Extracted mRNA was converted to cDNA by reverse transcription before real time quantitative PCR measurement of the housekeeping beta-actin gene. Plasma beta-actin mRNA at 2 hours (0.012; 0.0031-0.0297, median and range) was significantly lower (P= 0.022) than at 0 hours (0.12; 0.057-0.165) (P= 0.016). The levels decreased further at 5 hours (0.0037; 0.0024-0.011) (P= 0.004). The results show that plasma beta-actin mRNA levels decrease with time after blood collection and that this is likely to be due to degradation in vitro.

Constitutively active Akt is an important regulator of TRAIL sensitivity in prostate cancer.

TRAIL/Apo-2L is a member of the tumor necrosis factor superfamily and has recently been shown to induce apoptosis in cancer cells, but not in normal cells. In nude mice injected with human tumors, TRAIL reduces the size of these tumors without side effects. Akt promotes cell survival and block apoptosis. Some prostate cancer cells express high levels of Akt due to lack of active lipid phosphatase PTEN, a negative regulator of PI-3 kinase pathway, which may be responsible for drug resistance. The objective of this paper is to investigate the intracellular molecules that regulate TRAIL resistance. We have examined caspase-8 activity, BID cleavage, Akt activity, mitochondrial membrane potential (DeltaPsi(m)) and apoptosis in prostate cancer (LNCap, PC-3, PC-3M and DU145) cells treated with or without TRAIL. PC-3, PC-3M and DU145 cells are sensitive to TRAIL, whereas LNCap cells are resistant. LNCap cells express the highest level of constitutively active Akt, which is directly correlated with TRAIL resistance. TRAIL activates caspase-8 in all the cell lines. Downregulation of constitutively active Akt by PI-3 kinase inhibitors (wortmannin and LY-294002), dominant negative Akt or PTEN, renders LNCap cells sensitive to TRAIL. Inhibition of TRAIL sensitivity occurs at the level of BID cleavage. Inhibition of protein synthesis by cycloheximide also causes LNCap cells sensitive to TRAIL. Overexpression of Bcl-2 or Bcl-X(L) inhibits TRAIL-induced DeltaPsi(m) and apoptosis. Overexpression of constitutively active Akt in PC-3M cells (express very low levels of constitutively active Akt) restores TRAIL resistance. These data suggest that elevated Akt activity protects LNCap cells from TRAIL-induced apoptosis, and the PI-3 kinase/Akt pathway may inhibit apoptotic signals by inhibiting processing of BID. Thus, constitutively active Akt is an important regulator of TRAIL sensitivity in prostate cancer.

Pro-survival function of Akt/protein kinase B in prostate cancer cells. Relationship with TRAIL resistance.

Tumor necrosis factor superfamily member TRAIL/Apo-2L has recently been shown to induce apoptosis in transformed and cancer cells. Some prostate cancer cells express constitutively active Akt/protein kinase B due to a complete loss of lipid phosphatase PTEN gene, a negative regulator of phosphatidylinositol 3-kinase pathway. Constitutively active Akt promotes cellular survival and resistance to chemotherapy and radiation. We have recently noticed that some human prostate cancer cells are resistant to TRAIL. We therefore examined the intracellular mechanisms of cellular resistance to TRAIL. The cell lines expressing the highest level of constitutively active Akt were more resistant to undergo apoptosis by TRAIL than those expressing the lowest level. Down-regulation of constitutively active Akt by phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002, reversed cellular resistance to TRAIL. Treatment of resistant cells with cycloheximide (a protein synthesis inhibitor) rendered cells sensitive to TRAIL. Transfecting dominant negative Akt decreased Akt activity and increased TRAIL-induced apoptosis in cells with high Akt activity. Conversely, transfecting constitutively active Akt into cells with low Akt activity increased Akt activity and attenuated TRAIL-induced apoptosis. Inhibition of TRAIL sensitivity occurs at the level of BID cleavage, as caspase-8 activity was not affected. Enforced expression of anti-apoptotic protein Bcl-2 or Bcl-X(L) inhibited TRAIL-induced mitochondrial dysfunction and apoptosis. We therefore identify Akt as a constitutively active kinase that promotes survival of prostate cancer cells and demonstrate that modulation of Akt activity, by pharmacological or genetic approaches, alters the cellular responsiveness to TRAIL. Thus, TRAIL in combination with agents that down-regulate Akt activity can be used to treat prostate cancer.

Progressive renal disease: does the quality of the proteinuria matter or only the quantity?

Proteinuria is now accepted to be not just a sign of renal disease but also a contributory factor to the development of progressive tubulointerstitial fibrosis. Excellent correlations between the degree of proteinuria and rate of decline of glomerular filtration rate have been demonstrated. What has been investigated less is whether the type of protein found in the urine is important. Using transformed and primary human proximal tubular epithelial cells, we have investigated the binding of albumin and retinol binding protein to plasma membrane preparations and studied the response of the intact cells to increasing concentrations of these same proteins. We have preliminary evidence for differences in the pattern of binding of these two proteins to the plasma membrane receptors and also for differential release of pro-inflammatory cytokines from intact cells. These in vitro results, along with those of other groups, and some recent clinical findings suggest that the quality of proteinuria may play a role in the early development of interstitial fibrosis. Furthermore, the use of such in vitro model systems based on human proximal epithelial cell culture can provide a means of evaluating the potential significance of different markers of tubular damage.

Reference ranges for plasma cystatin C and creatinine measurements in premature infants, neonates, and older children.

AIM: To establish a reference range in the paediatric population for the new glomerular filtration rate (GFR) marker, cystatin C, and to compare it with that of creatinine. METHODS: Cystatin C and creatinine were measured by particle enhanced nephelometric immunoassay (PENIA) and fixed interval Jaff¿e methods, respectively, in 291 children aged 1 day to 17 years, including 30 premature infants with gestational ages ranging from 24 to 36 weeks. RESULTS: In the premature infants, concentrations of both cystatin C and creatinine were significantly raised compared with term infants, with cystatin C concentrations being between 1.10 and 2.06 mg/litre and creatinine between 32 and 135 micromol/litre. In premature infants, there was no significant relation between gestational age and cystatin C or creatinine concentration. Creatinine concentrations fell to a nadir at 4 months of age, rising gradually to adult values by about 15-17 years of age, in contrast to cystatin C, which fell to a mean concentration of 0.80 mg/litre by the 1st year of life, and remained constant throughout adulthood up to the age of 50 years. Neither analyte showed any influence of sex. CONCLUSION: The measurement of cystatin C, rather than creatinine, is more practical for monitoring GFR changes in the paediatric population.

The use of optical sensors to understand cellular interactions with renal cells.

Optical biosensor technology has revolutionized the assessment of receptor binding, enabling the characterization of low affinity interactions in real time. We report the application of the LAsys Optical Biosensor to the investigation of the affinity and specificity of the putative proximal tubular scavenging receptor for protein reabsorption and the specificity of AGE-modified protein interactions with primary human mesangial cells. Using the LLCPK cell line, the carboxy-methyl dextran cuvette surface and five different proteins (ranging in size and charge), we have shown that there is evidence to support the existence of a single scavenging receptor for all the proteins tested. The proteins competed with each other differing only in their relative binding affinity for the common receptor. We have also shown that human mesangial cells can bind to AGE-modified human serum albumin (AGE-HSA) immobilized onto the carboxylate surfaced planar cuvette and that binding can be inhibited using increasing concentrations of soluble AGE-HSA. However, increasing concentrations of soluble Non-AGE modified HSA can also inhibit binding to a similar extent which implies that there is relatively little AGE-receptor (RAGE) expression on cultured primary human mesangial cells. These results demonstrate the exciting potential of this technology as a tool to explore cellular interactions with renal cells.

Measurement of the kinetics of protein uptake by proximal tubular cells using an optical biosensor.

BACKGROUND: The affinity and specificity of protein reabsorption by proximal tubular cells have been investigated using techniques for monitoring endocytosis, demonstrating a high capacity but low affinity process. It is not known whether uptake is through binding to a single binding site/receptor with differing affinities, or if there are several classes of binding sites receptors, each specific for differing proteins or groups, such as, high or low molecular weight proteins. METHODS: We have developed a novel technique for analyzing the kinetics of protein binding to tubular cells using a optical biosensor system. We have studied the binding of cultured LLCPK cells to albumin and RBP immobilized onto the sensor. By adding increasing concentrations of competing proteins [varying in molecular weight from 66,000 to 11,800 D and pI from 4.6 to 9.2 as represented by albumin, alpha1-microglobulin (alpha1M), retinol binding protein (RBP), cystatin C and beta2-microglobulin (beta2m)], specific and inhibitable cell binding was demonstrated. RESULTS: Equilibrium constants, KA, could be calculated from the reciprocal of the protein concentration causing 50% inhibition in binding rate. These were: albumin = 8.0 x 10(4) M(-1), alpha1M = 2.0 x 10(5) M(-1), RBP = 2.7 x 10(4) M(-1), cystatin C = 2.0 x 10(4) M(-1), beta2m = 4.2 x 10(3) M(-1). There were no significant differences between the measured KA's whether RBP or albumin were immobilized on the surface. CONCLUSIONS: All the proteins gave similar shaped inhibition profiles, suggesting that there is one binding site/receptor for all proteins studied, regardless of molecular weight or charge, but there are differing affinities for each protein.

Development and validation of an automated latex-enhanced immunoassay for prealbumin.

The measurement of circulating prealbumin has been shown to be clinically useful in the assessment of nutritional status, both as an initial screen and in the monitoring of nutritional recovery. We describe a fully automated, noncompetitive, homogeneous, light-scattering immunoassay that has been developed for this analyte on a Dimension (Dade) analyzer. A sheep anti-prealbumin IgG fraction was covalently coupled to 40-nm chloromethyl styrene particles and, after the addition of sample, polyethylene glycol-assisted immunoagglutination was monitored by turbidimetry. The prealbumin working assay range was 8-550 mg/L at a sample volume of 2 microL and a reaction time of 6.5 min. When data were analyzed using ANOVA, total and within-run assay imprecision values (CVs) were 1-5%, and calibration and reagent stabilities were in excess of 40 days. Mean analytical recoveries were 102% +/- 4% (SD), and there was no lack of parallelism. Hemolysis, lipemia, and bilirubin did not interfere. Both plasma anticoagulated with heparin or EDTA and serum from plain or serum-separation tubes were acceptable as sample matrices. Comparison with the Beckman Array method gave a Passing and Bablok regression of: Dimension analyzer = 1.01Beckman + 7.1 (n = 103), using a common calibrator. We conclude that the prealbumin method is appropriate for clinical use according to the analytical criteria used in this study.

Measurement of albumin and low molecular weight proteins in the urine of newborn infants using a cotton wool ball collection method.

OBJECTIVE: The aim of this study was to investigate the inter-relationship between urinary excretion of alpha-1-microglobulin (A1M), retinol-binding protein (RBP) and albumin in term and premature neonates, with urine collected into cotton wool balls and extracted by a novel method. SUBJECTS AND METHODS: Sixty-four infants were studied on the first day of life; 26 had been born at term (37-42 weeks gestation) and 38 prematurely (24-28 weeks n = 16, 29-36 weeks n = 22). Urine collected into cotton wool balls was analysed following a new detergent extraction method, which resulted in a recovery rate of 94-107% for albumin. A1M, RBP and creatinine. RESULTS: Urinary protein excretion, expressed as a ratio to urinary creatinine, decreased significantly with increasing gestational age (24-28 weeks, 29-36 weeks, 37-42 weeks: albumin:creatinine ratio mg/mmol mean 96.9, 31.7, 19.3; A1M:creatinine ratio mg/mmol mean 99.3, 37.0, 7.8; RBP:creatinine ratio mg/mmol mean 16.2, 3.8, and < 0.01, below the limit of detection, respectively). When results were corrected for birthweight, this gestation-associated effect was still present for A1M and RBP, but not for albumin. In premature infants there was a significant positive correlation between A1M:creatinine ratio and RBP:creatinine ratio (r = 0.85), and also between albumin and both A1M and RBP (r = 0.82 and 0.77). CONCLUSION: Increased excretion of A1M, RBP and albumin at earlier gestational ages is probably due to proximal tubular immaturity, although tubular damage and also glomerular dysfunction cannot be excluded as possible explanations.

A simple, automated, nonextraction assay for serum digoxin using particle-enhanced immunoassay.

We have adapted a latex particle-enhanced immunoassay for serum digoxin to a centrifugal analyser. A 4-microliter serum sample (without pretreatment) inhibits the monoclonal antibody induced aggregation of digoxin-coated latex particles. The total assay time is 10 min and mean analytical recoveries were 98.1%. Intra and interassay precision were < 4.2 and < 15.0%, respectively. Method comparison with an established fluorescence polarisation immunoassay (FPIA) gave R = 0.97, and a Deming regression analysis of particle-enhanced turbidimetric inhibition immunoassay (PETINIA) = FPIA x 0.78 + 0.007 microgram/L (n = 91). There was no evidence of significant interference from digoxin-like immunoreactive compounds in patients with chronic renal failure. This assay can be adapted to most photometric analysers used in routine laboratories and is a significant advance in the sensitivity of latex particle-enhanced immunoassays in a serum matrix.

Development and validation of a particle-enhanced turbidimetric inhibition assay for urine albumin on the Dade aca analyzer.

The measurement of urine albumin now has a well-established role in the monitoring of patients with diabetes mellitus. We have developed a particle-enhanced immunoturbidimetric inhibition assay for urine albumin on the Dade aca analyzer. The inhibition approach removes any of the potential antigen excess difficulties that could be expected from the wide clinical range of urine albumin, but retains the sensitivity advantages of latex-enhanced immunoturbidimetry. Human serum albumin (HSA) is covalently attached to 40-nm poly(chloromethyl)styrene-modified latex particles. This reagent, along with monoclonal antibody to HSA, is aliquoted into the aca reagent pack along with polyethylene glycol 8000 in a tablet form (giving a final reaction concentration of 15 g/L). A 150 mmol/L phosphate buffer, pH 7.8, is used to fill the reagent pack in the instrument and the agglutination reaction is monitored at 340 nm. The sample volume is 100 microL and the calibration curve covers the range 2-250 mg/L. Evaluation of commercial scale reagents against the Beckman Array nephelometric immunoassay system gave a Deming regression correlation of aca = 0.87 x Beckman + 8.5, r = 0.995, n = 145. Mean analytical recovery was 104+/-4.5%, n = 20, and there was no evidence of a lack of parallelism. Interassay precision was 8.8% at 10.0 mg/L and <2.5% at >65 mg/L. Calibrator stability was in excess of 60 days. A small reference range study (24-h urine collections, n = 27) gave a mean of 5.6 mg/L with a range of 0.5-16.2 mg/L. Analytical sensitivity (2.5 SD from zero) was 0.40 mg/L.