A reliable and easy to transport quality control method for chlamydia and gonorrhoea molecular point of care testing.

Quality control (QC) is an essential component of point-of-care testing programs. In the context of a randomised-controlled trial (TTANGO) using GeneXpert (Xpert) Chlamydia trachomatis and Neisseria gonorrhoeae (CT/NG) point-of-care testing in remote areas of Australia, we aimed to develop and utilise a stable positive control material. Bacterial cultures of CT and NG were resuspended together to provide cycle threshold (Ct) values of approximately 25 cycles for both CT and NG when tested on the Xpert CT/NG assay. These positive control suspensions were dried in aliquots, heat inactivated, and then provided to 12 participating health services as research-only QC samples in kit form. At each service, a QC sample was resuspended and tested each month on the Xpert. QC results, including Xpert Ct values, were analysed from each site over 30 months and we calculated costs per QC sample. Overall, at 12 health services there were 89 QC samples tested (average of 8 tests per site per year). Mean Ct values for the 89 controls samples were 25.25 cycles (SD = 1.15) for CT, 24.04 cycles (SD = 1.400) for one NG target and 23.35 cycles (SD = 1.55) for the other NG target. No significant differences in Ct value for CT or NG controls were observed over a trial period of 30 months. Positive QC samples for research use in a trial of a molecular point-of-care assay were inexpensive to produce and stable when stored at 2-8°C. For routine use, additional requirements such as meeting National Association of Testing Authority (NATA) regulations and Therapeutic Goods Administration (TGA) approval will need to be achieved.

Results of an Aboriginal community-based renal disease management program incorporating point of care testing for urine albumin:creatinine ratio.

INTRODUCTION: There has been a significant increase in the burden of renal disease among Aboriginal Australians over the past 15 years. Urine albumin:creatinine ratio (ACR) is a well-established marker of microalbuminuria and can be conveniently performed on the DCA 2000 point-of-care testing (POCT) analyser (Bayer Australia; Melbourne, VIC, Australia) with an on-site result available in 7 min. The application of the urine ACR POCT for renal disease risk assessment was pioneered by our group in the Umoona Kidney Project. This article describes the results of the management arm of the Umoona Kidney Project, which used point-of-care urine ACR testing for the first time within a management framework to monitor albuminuria in patients at highest risk of renal disease. The article also examines the analytical quality of POCT results and overall community acceptance of the Umoona Kidney Project. METHODS: Adults clinically assessed by Flinders Medical Centre renal specialists as being at greatest risk for renal disease were offered the ACE inhibitor (ACEI) perindopril on a voluntary basis. Selected renal markers, including POCT urine ACR (conducted on-site by Umoona's Aboriginal health worker team), plasma electrolytes, urea, creatinine, calculated glomerular filtration rate and blood pressure were measured six monthly. Regular quality control testing was undertaken to monitor the analytical performance of the POCT analyser. A culturally appropriate questionnaire was designed and implemented to assess community satisfaction with the project. RESULTS: In all, 231 patient management consultations were conducted over a two year period, with over 70% of patients having four or more (up to a maximum of eight) consultations; 35 patients (mean age 49.2 [+/-2.3] years, 54% males) participated voluntarily in the management arm. All were overtly hypertensive, hypertensive with other risk factors or had diabetes. The renal status of these patients was followed for a mean of 63 +/- 4.5 weeks. In total, 111 POCT urine ACR tests were performed for patient management (mean 3.2 tests per patient). There was no significant difference in POCT urine ACR in the study period with a median (and inter-quartile range) of 5.7 mg/mmol (1.2-15.2) pre-ACEI and 4.3 mg/mmol (1.3-16.7) post-ACEI treatment (p = 0.50, Wilcoxon signed ranks test). The calculated glomerular filtration rate altered from 110 to 118 mL/min (p = 0.019, paired t-test). There was no change in the group plasma potassium, urea and creatinine. Collectively these results indicate a stabilisation in renal function among the management group. Blood pressure (both lying and standing) fell significantly in the study period. The imprecision for urine ACR quality control POCT conducted during the management program was within nationally and internationally accepted precision goals for urine albumin, creatinine and ACR. Fifty community members completed the satisfaction questionnaire. Three-quarters of respondents felt there were no cultural barriers in providing a urine sample for urine ACR POCT. CONCLUSIONS: The management arm of the Umoona Kidney Project was effective in stabilising the renal function and improving the blood pressure of community members identified to be at greatest risk of kidney disease. POCT urine ACR testing can be utilised, not only for community risk assessment, but also for patient management. The Umoona Kidney Project was well accepted by the health service and community members.

An innovative Australian point-of-care model for urine albumin: creatinine ratio testing that supports diabetes management in indigenous medical services and has international application.

BACKGROUND: Type 2 diabetes is the leading cause of end-stage renal failure in Australia's indigenous people. The measurement of urine albumin:creatinine ratio (ACR) as a marker for early renal disease is an important component of the management of indigenous patients with diabetes. METHODS: An innovative national program (Quality Assurance for Aboriginal Medical Services [QAAMS]) for point-of-care (POC) urine ACR testing on the DCA 2000 analyser (Bayer Diagnostics) was established to monitor microalbuminuria in indigenous people with diabetes in 30 Aboriginal and Torres Strait Islander medical services across Australia. Aboriginal health workers perform the ACR test. The QAAMS model provides ongoing education and training, an annual workshop, monthly quality assurance testing and a telephone help hotline. Quality assurance testing is conducted using paired, linearly related samples with a wide range of ACR concentrations (1-25 mg/mmol). RESULTS: The average participation rate across four six-monthly QAAMS ACR testing cycles was 83%. In all, 94% of 1163 quality assurance tests performed were within the preset limits of acceptability. The median precision (coefficient of variation percent for ACR quality assurance testing averaged 5.4%, well within desirable performance specifications. Between-site accuracy was excellent. CONCLUSION: This unique POC model for supporting diabetes management is the first of its type to be developed for indigenous communities and has considerable potential to be adopted worldwide.

Measurement of plasma LDL cholesterol in patients with diabetes.

OBJECTIVE: To assess the accuracy of plasma LDL cholesterol concentrations estimated by the Friedewald formula and a direct immunoseparation method by comparison with a reference ultracentrifugation procedure in patients with diabetes. RESEARCH DESIGN AND METHODS: Fasting plasma samples with triglyceride concentrations < 4.5 mmol/l were collected from 100 patients with diabetes (28 type I and 72 type II) and LDL cholesterol concentrations were compared by the three methods. RESULTS: LDL cholesterol values determined by the reference beta-quantitation procedure were highly correlated with both the Friedewald formula (r = 0.96) and a direct immunoseparation method (r = 0.92). Calculated (Friedewald) LDL cholesterol coincided with the reference method with < 10% error in 74% of the total diabetic group (82% of type I and 68% of type II diabetic patients). However, agreement between the direct LDL cholesterol and reference methods was significantly less (P = 0.02), with only 44% of patients having an error of < 10% (52% of type I and 41% of type II diabetic patients). The direct immunoseparation method for LDL cholesterol showed a positive bias with increasing triglyceride concentrations, particularly for patients with type II diabetes. CONCLUSIONS: In the group of diabetic patients studied with plasma triglyceride concentrations < 4.5 mmol/l, the Friedewald formula provided an accurate estimation of LDL cholesterol. The direct immunoseparation method significantly overestimated LDL cholesterol at triglyceride levels between 2 and 4.5 mmol/l.

Quantitative method for determining serum alkaline phosphatase isoenzyme activity I. Guanidine hydrochloride: new reagent for selectively inhibiting major serum isoenzymes of alkaline phosphatase.

The potential use of the protein denaturant guanidine hydrochloride to inhibit selectively the enzyme activity of serum alkaline phosphatase isoenzymes from liver, bone, intestine, and placenta was investigated. Inhibition of each isoenzyme was shown to be dependent on time and concentration of inhibitor. In the presence of 0.3 mol/l (28.7 g/l) guanidine hydrochloride for 170 seconds 14%, 47%, and 90% of the total alkaline phosphatase activity remained in samples of bone, liver, and intestinal origins, respectively. In contrast, the activity of the placental isoenzyme increased by 24%. The degree of inhibition was shown to be independent of total alkaline phosphatase activity. Investigations were performed at 37 degrees C using the Cobas Bio centrifugal analyser. We conclude that this reagent has several practical advantages over urea as a selective inhibitor of alkaline phosphatase isoenzymes, including a faster and more reproducible inhibition at a much lower reagent concentration.

Evaluation of an enzymatic method for determining creatinine in plasma.

An enzymatic kit method for the determination of plasma creatinine was optimised for use with a centrifugal analyser and its performance characteristics and practicability compared with an end point and a kinetic Jaffé-based method. The enzymatic method exhibited several advantages over Jaffé-based methods--namely, smaller sample size, rapid sample throughput (200 per hour), and improved specificity. Glucose, acetoacetate, and cefoxitin did not interfere with the enzymatic method, although bilirubin did cause a negative interference which depended on both creatinine and bilirubin concentrations. The enzymatic method has particular clinical application in neonates, diabetic ketotic patients, and those receiving cephalosporins.

Evolution of a national urine quality assurance programme: the Australasian experience, 1981-1983.

A national interlaboratory quality assurance programme for quantitative urine analysis has been conducted over the past three years in Australasia under the auspices of the Royal College of Pathologists of Australasia and the Australian Association of Clinical Biochemists. Analysis of urine calcium has consistently improved over the three year period whereas urine protein analysis has consistently declined. Based on the findings in 1983, it is considered that urine sodium, potassium, creatinine, phosphate, glucose, and chloride are currently being measured satisfactorily by Australasian laboratories, while the analyses of urine proteins, urate, oxalate, 5-hydroxyindoleacetic acid and 4-hydroxy-3-methoxymandelic acid still require substantial improvement.

Quantitative method for determining serum alkaline phosphatase isoenzyme activity II. Development and clinical application of method for measuring four serum alkaline phosphatase isoenzymes.

A method for quantitating the liver, bone, intestinal and placental alkaline phosphatase activity of serum, using an algorithm for converting selective inactivation by guanidine hydrochloride, L-phenylalanine, and heat into equivalent isoenzyme activity is described. The method can individually quantify mixtures of isoenzymes to within a margin of 3%; it has acceptable reproducibility and has been used to develop both age and sex related reference ranges. Analysis time is about 30 minutes. The clinical reliability of this method has been shown in a study of 101 patients, in 79% of whom isoenzyme results were compatible with the final clinical diagnosis; in 10% a clinical diagnosis resulted from isoenzyme analysis, and in a further 11% the source of the increased alkaline phosphatase activity was identified and supported by electrophoresis, with a definite clinical diagnosis yet to be made.

Adaptation of methods for glutamate dehydrogenase and alcohol dehydrogenase activities to a centrifugal analyser: assessment of their clinical use in anoxic states of the liver.

Sensitive, precise, and rapid methods for the measurement of alcohol dehydrogenase (ADH) and glutamate dehydrogenase (GDH) were developed on the Cobas Bio centrifugal analyser. The optimal pH for ADH in caucasians was 9.8. Non-linearity of ADH enzyme activity was observed when samples were diluted in saline; linearity was restored when inactivated serum was used as diluent. ADH was shown to be a sensitive index of liver anoxia due to cardiorespiratory disturbance (clinical sensitivity 90%) and generalised anoxia. GDH exhibited sensitivity equal to that of alanine aminotransferase (ALT) but was inferior to gamma-glutamyltransferase (GGT) in the detection of specific liver disease. Both ADH and GDH were sensitive indicators of alcoholic liver disease.

Lipoprotein profiling by high performance gel chromatography.

High performance gel chromatography (HPGC) was used to separate lipoproteins on the basis of their size and to generate lipoprotein profiles for plasma collected from patients with different lipoprotein phenotypes. These profiles provided a direct measurement of low density lipoprotein (LDL)-cholesterol which was more precise than LDL-cholesterol values calculated by the Friedewald equation. In addition, LDL-cholesterol concentrations were obtained in patients with combined hyperlipidemia in whom LDL-cholesterol could not be accurately calculated by the Friedewald equation. The response of LDL-cholesterol to the drug gemfibrozil was reliably monitored and in addition changes in LDL particle size could be assessed from the LDL apolipoprotein B/cholesterol ratio. HPGC also assisted in the diagnosis of type III hyperlipidemia by revealing a characteristic lipoprotein profile. HPGC-derived lipoprotein profiles provided additional useful clinical information for combined hyperlipidemia (Fredrickson lipoprotein phenotypes IIb, III).

A quality assurance programme for quantitative urine analyses.

Eighty-six Australasian laboratories participated in an interlaboratory quality assurance programme for 10 urine analytes. Twelve liquid samples were prepared from commercial lyophilized urine control material and distributed in 3 batches of 4. Use of pre-set acceptability limits for total laboratory error and target values facilitated timely feedback in graphic form. The samples had concentrations which were linearly related; this allowed simple calculation of overall imprecision and bias, graphic feedback of all submitted results, and comparison of performance between laboratories. A number of unsuitable and poor methods were identified. Particular attention must be paid, in future, to more widespread use of appropriate calibration and quality control materials, to avoidance of transcription and calculation errors, and to analysis of urine samples with elevated levels of analyte. Current laboratory performance can meet analytical goals for analyses of urine creatinine, phosphate, urate, and glucose but analysis of urine sodium, potassium, urea, calcium, osmolality, and proteins require a significant improvement.

An inter-laboratory survey of qualitative urinalysis.

An inter-laboratory survey of qualitative urinalysis was carried out in Australasia during 1981. Eighty-one laboratories analysed 6 samples of urine distributed in 3 batches of 2 at regular bimonthly intervals, mostly with commercially available reagent strips. Fewer than 30% of laboratories performed any form of quality control for analytes other than pH. Results indicated that improvement in the analysis of urine bilirubin, protein, glucose, ketones and blood is required, and recommendations to improve standards are made.

Nephelometric determination of total protein in cerebrospinal fluid and urine using benzalkonium chloride as precipitation reagent.

Four different protein precipitants, namely trichloroacetic acid, sulphosalicylic acid, benzethonium chloride and benzalkonium chloride, were used to estimate the total protein concentration in cerebrospinal fluid and urine by nephelometry. Protein determinations for 50 cerebrospinal fluid samples and 100 urine samples were compared with values obtained by a trichloroacetic acid-Ponceau S spectrophotometric method. All methods were correlated well, but total protein results using acid precipitants were usually lower, while results obtained using benzethonium chloride were generally higher, than results obtained by the trichloroacetic acid-Ponceau S method. Anomalous results were obtained for some urine samples with benzethonium chloride, but not with benzalkonium chloride. Assays using benzalkonium chloride as precipitating reagent showed good precision and closest agreement with the trichloroacetic acid-Ponceau S dye-binding method. The use of benzalkonium chloride as precipitant is recommended for automated cerebrospinal fluid and urine protein estimations by nephelometry.

Variation in plasma apolipoprotein A-1 and B concentrations following myocardial infarction.

Measurements of plasma apolipoprotein A-1 and B concentrations are increasingly used for the laboratory assessment of risk of coronary artery disease (CAD). This study of 22 patients investigated the response of plasma apolipoprotein A-1 and B levels for up to 20 days following a myocardial infarction. Seven of these patients participated in a clinical trial using the drug Tissue Plasminogen Activator (TPA). We established that, unlike many other plasma proteins, apolipoproteins do not display a classic acute phase response following myocardial infarction, although large variations in plasma apolipoprotein levels were observed in the patients investigated. Our studies also show that the measurement of plasma apolipoproteins A-1 and B to assess future CAD risk in myocardial infarction patients should be deferred for a minimum of at least 14 days post-infarction. No significant difference was observed in the pattern of apolipoprotein response between patients receiving TPA and those not given this drug.

Albuminuria in a remote South Australian Aboriginal community: results of a community-based screening program for renal disease.

INTRODUCTION: The poverty, poor environmental living conditions and poor health standards experienced by Aboriginal Australians in some communities in rural and remote Australia have been described recently as 'fourth world'. For more than a century Aboriginal people have suffered the effects of dispossession of their land; destruction of their traditional culture and values; and exposure to infectious diseases, alcohol and the Western diet that is high in fat and sugar. Collectively these factors have contributed to the prevalence of chronic disease that afflicts Aboriginal people. In particular, renal disease has emerged during the last decade as a major contemporary health problem for Aboriginal Australians. According to the latest age- and sex-adjusted figures, Aboriginal people now have approximately nine-fold the risk of non-Aboriginal Australians of developing end-stage renal disease. In parts of Australia's Northern Territory, where Aboriginal people represent over 20% of the Territory's population, the rates of end-stage renal disease have been described as 'epidemic', reaching 2700 per million in the Tiwi Islands. In response to a request from the Umoona Tjutagku Health Service in mid 1997, the Renal Unit at Flinders Medical Centre, Adelaide, South Australia, formed a partnership with the health service to conduct a renal-disease screening program for adult members of the Umoona Community at Coober Pedy, a town 850 kilometres north of Adelaide. The partnership was later expanded to include screening for children (conducted by the Renal Unit at the Women's and Children's Hospital, Adelaide, South Australia). The community named the program 'The Umoona Kidney Project'. The Umoona community had recently experienced the dislocation of a number of its older people who suffered from advanced renal disease and were undergoing dialysis in a variety of centres in South Australia and the Northern Territory. As a result, the community had suffered social trauma. Consistent with the community's overall holistic approach to healthcare, the community wanted the renal program to provide a focus for community awareness of and knowledge about chronic disease, as well as to complement existing health programs. OBJECTIVES: The study objectives were to identify the prevalence of risk factors for renal disease, notably albuminuria, in adults from a remote Aboriginal community, and to examine the association of albuminuria with other risk factors; to empower Aboriginal health workers to self-manage a sustainable, community-controlled renal health program; and to assess the reliability and cultural acceptability of point-of-care technology for detecting renal disease. METHOD: The study was a three-year cross-sectional voluntary adult screening program (The Umoona Kidney Project). The study was performed as a partnership between the Flinders Medical Centre Renal Unit and the Umoona Tjutagku Health Service, and it involved nephrologists, medical scientists, Aboriginal health workers and clinical nurses. SETTING: Umoona Tjutagku Health Service, 850 km north of Adelaide. PARTICIPANTS: 158 adult members of the Umoona community: 58 males (37%; mean age = 43.8 years, range 23-78) and 100 females (63%; mean age = 39.6 years, range 18-72). MAIN OUTCOME MEASURES: First morning urine albumin : creatinine ratio measured by the Bayer DCA 2000 point-of-care analyser machine (Bayer Australia, Melbourne, Australia); lying and standing blood pressure; random blood glucose; body mass index; urinalysis. RESULTS: The study found that of screened adults, 29/149 (19%, 95% C.I. 13%-27%) had persistent microalbuminuria and 13/149 (9%, 95% C.I. 4%-14%) had persistent macroalbuminuria; 62/148 participants (42%, 95% C.I. 34%-50%) had overt hypertension; 35/145 participants (24%, 95% C.I. 17%-32%) had diabetes; 3 participants were newly diagnosed as having non-insulin dependent diabetes; 96/148 participants (65%, 95% C.I. 57%-73%) were either overweight or obese. Strong correlation was observed between the progression of albuminuria and age, all blood pressure categories, blood glucose, body mass index and an increasing number of risk factors. CONCLUSIONS: The Umoona Kidney Project identified a significant community burden of previously unknown incipient and established renal disease that required addressing via clinical- and community-based interventions. The DCA 2000 was a reliable instrument for detecting albuminuria on-site in the remote clinical location and was well accepted by Aboriginal health workers and community participants.

Falsely low estimation of triglycerides in lipemic plasma by the enzymatic triglyceride method with modified Trinder's chromogen.

The enzymatic assay of triglyceride, based on the use of L-glycerol-3-phosphate oxidase (EC 1.1.3.21) and a modified Trinder's chromogen involving 4-chlorophenol, is subject to strong negative interference at concentrations of triglyceride greater than 20 mmol/L, such as occur in grossly lipemic plasma. This interference is caused by the rapid utilization of oxygen, resulting in the reaction becoming transiently anaerobic. The dye product already formed may then be reduced ("bleached") by acting as an alternative electron acceptor for glycerol-3-phosphate oxidase. Reduction of the dye leads to a marked decrease in final absorbance at 505 nm. Grossly underestimated values for triglyceride concentrations, apparently within the linear range of the assay, may therefore be inadvertently obtained with equilibrium methods. We suggest that samples giving unexpectedly low results for lipemic plasma should be re-assayed after dilution or with use of a smaller volume of sample.