Evaluation of the ARKRAY AUTION Eleven reflectometer in detecting microalbuminuria with AUTION Screen test strips and proteinuria with AUTION Sticks 10PA strips.

OBJECTIVE: Albumin/creatinine and protein/creatinine ratios were measured with the ARKRAY AUTION Eleven reflectometer using AUTION Screen and AUTION Sticks 10PA strips, respectively, against quantitative Siemens Advia reference procedures from 368 patient urines, as an evaluation of their applicability for use in points-of-care and small laboratories. MATERIAL AND METHODS: Direct reflectance measurements were utilized to estimate imprecision, as well as to suggest reclassification of ordinal scale categories into normoalbuminuria, microalbuminuria and macroalbuminuria groups (3.4 g/mol and 34 g/mol cut-off limits, corresponding to 30 mg/g and 300 mg/g creatinine in conventional units). RESULTS: Analytically, ordinal scale albumin/creatinine ratios agreed in 86% of cases with those obtained from Advia measurements, resulting in a kappa coefficient of 0.79. Protein/creatinine ratios of the AUTION Sticks 10PA strip were classified into three groups at limits of 11.3 g/mol and 56.6 g/mol (100 mg/g and 500 mg/g in conventional units), with an agreement of 77% and a kappa coefficient of 0.65 against Advia procedures. To optimize clinical outcomes, cut-off reflectances of ordinal scale categories of AUTION Eleven were adjusted. The clinical specificity of detecting an increased albumin/creatinine ratio was then increased from 81% to 95%, with clinical sensitivity kept at 88% at the 3.4 g/mol limit of the reference procedure. Clinical specificity of the albuminuria field alone (at a clinical sensitivity of 88%) was only 73%. Adjustments to cut-off reflectances of the reported categories for protein/creatinine ratios increased clinical specificity from 54% to 94%, while losing clinical sensitivity from 97% to 89% only, with an improved concordance of 83% and a kappa coefficient of 0.75 against Advia measurements. The combination to creatinine measurements improved clinical specificity compared to 50% by the protein field alone. In economic terms, it is estimated that population screening for microalbuminuria using the AUTION Eleven reflectometer is cheaper than by quantitative albumin/creatinine measurements alone, based on the incidence of end-stage renal disease of 90 patients/million/year at the Northern Ostrobothnia Hospital District.

Pre-analytical factors and measurement uncertainty.

Pre-analytical factors are an important source of variation or errors in clinical laboratory measurements. Based on the new accreditation standards, medical and laboratory professions now seek to develop tools to deal systematically with these diverse factors. Several obvious pre-analytical uncertainty components were estimated in pragmatic experiments and combined with data on analytical variation and literature knowledge on biological variation, to estimate the measurement uncertainty of most common chemical and haematological examinations in clinical laboratories. The main aim was to assess quality specifications for regional laboratory services. The expanded measurement uncertainties (level of confidence 95%) of serum cholesterol, albumin and potassium remained within 13-16%. The major uncertainty component for cholesterol was biological variation, whereas those for albumin and potassium were sample collection and pretreatment. The measurement uncertainties for serum free thyroxin, thyrotropin and C-reactive protein, 20%, 42% and 125% respectively, were largely due to their biological variation. The measurement uncertainties of basic erythrocyte parameters (erythrocyte count and mean corpuscular volume, blood haemoglobin concentration) were less than 10%. Larger measurement uncertainties were obtained for thrombocyte and leukocyte counts, 24 and 31%, respectively, and for the reticulocyte fraction, 41%.

Reference intervals for the markers of proteinuria with a standardised bed-rest collection of urine.

Reference intervals for markers of proteinuria or glomerular charge selectivity were measured in 61 healthy female and 61 healthy male individuals. Timed bed-rest and daytime collections were used to assess significance of preanalytical variability of results. Bed-rest collections are advisable for research on renal damage, whereas in routine care, robust protein/creatinine ratios work as practical estimates of protein excretion rates, the correlations to excretion rates improving with increasing proteinuria. For glomerular charge selectivity, pancreatic/salivary isoamylase clearance ratio showed lower within-subject biological variation than IgG/IgG4 clearance ratio, allowing more accurate classification into normal and reduced charge selectivity. With our method, the lower 2.5% reference intervals for isoamylase clearance ratio were 1.1 in men and 1.9 in women.

Reference intervals for cystatin C in pre- and full-term infants and children.

Cystatin C is a non-glycated, 13-kDa basic protein produced by all nucleated cells. Recent studies have indicated that the plasma concentration of cystatin C is a better marker for glomerular filtration rate (GFR) than plasma creatinine, which is most commonly used for this purpose. We established reference values for plasma cystatin C in pre- or full-term infants and children. For comparison we also measured the creatinine concentration in the same samples. Cystatin C was measured by a commercially available immunoturbidimetric method with a Hitachi 704 analyzer in sera obtained from 58 pre-term infants, 50 full-term infants and 299 older children (132 girls, 167 boys, median age 4.17 years, range 8 days to 16 years). No sex differences were found. The pre-term infants had higher cystatin C concentrations (mean 1.88 mg/l, SD 0.36 mg/l) than the full-term (mean 1.70 mg/l, SD 0.26 mg/l, P=0.0145). The reference interval for pre-term infants calculated non-parametrically was 1.34-2.57 mg/l and for full-term infants 1.36-2.23 mg/l. The cystatin C concentration decreased rapidly after birth, and above 3 years of age did not depend on age. The reference interval for children 3-16 years of age calculated non-parametrically was 0.51-1.31 mg/l. Younger children (<1 year: 0.75-1.87 mg/l; 1-3 years: 0.68-1.60 mg/l) had slightly, but significantly, higher plasma cystatin C levels.

The role of automated urine particle flow cytometry in clinical practice.

Urine particle flow cytometers (UFC) have improved count precision and accuracy compared to visual microscopy and offer significant labor saving. The absence of an internationally recognized reference measurement procedure, however, is a serious drawback to their validation. Chamber counting by phase contrast microscopy of supravitally-stained uncentrifuged urine is considered the best candidate for reference. The UF-100 (Sysmex Corporation, Japan) identifies RBC, WBC, squamous epithelial cells, transitional epithelial and renal tubular cells (SRC), bacteria, hyaline and inclusional casts, yeast-like cells, crystals and spermatozoa, using argon laser flow cytometry. Evaluations have established acceptable linearity over useful working ranges, with an imprecision that is consistently and significantly less than microscopy, and with negligible carry-over. Comparisons of UFC with chamber counts, quantitative urine microscopy, sediment counts, test strips, bacterial culture and urine density are reviewed. Clinical studies include diagnosis and monitoring of urinary tract infection; localization of the sites of hematuria; and diagnosis, monitoring and exclusion of renal disease. The most popular approach is to combine test strips with UFC for primary screening either always by both methods or by using test strips for analytes unrelated to particles analyzed by UFC. Expert systems now exist combining both test modalities based on user definable decision rules. The implementation of such a strategy significantly reduces microscopy review and saves time and expense without diminishing clinical utility.

Renal function and blood pressure five years after puumala virus-induced nephropathy.

BACKGROUND: Nephropathia epidemica (NE) is a mild form of hemorrhagic fever with renal syndrome caused by Puumala hantavirus. Its long-term prognosis is considered favorable. Some reports suggest, however, that a previous hantavirus infection increases the risk of hypertension. METHODS: We studied 46 previously healthy subjects (26 males and 20 females, mean age of 44 years) who had serologically confirmed NE three to seven years previously, and 38 healthy, seronegative controls (22 males and 16 females, mean age of 44 years). Ambulatory blood pressure (ABP) was monitored. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were determined by 51CrEDTA and 131I-hippurate clearances, respectively. The filtration fraction (FF) was calculated. Quantitative 24-hour urinary protein excretion (UprotE) and timed overnight urinary excretion of alpha1-microglobulin were measured. RESULTS: The NE patients had a higher mean ambulatory systolic BP than the controls (123 +/- 13 vs. 117 +/- 9 mm Hg, P = 0. 008). GFR and FF were increased in patients compared with controls (GFR, 120 +/- 20 vs. 109 +/- 14 mL/min/1.73 m2, P = 0.006; FF, 19 +/- 3 vs. 18 +/- 3%, P = 0.030), but ERPF did not differ between the groups. The patients also had higher UPE than the controls (median 0. 18 g/day, range 0.12 to 0.38 vs. median 0.14 g/day, range 0.09 to 0. 24, P < 0.001, respectively). The overnight urinary excretion rate of alpha1-microglobulin exceeded 7 microg/min in nine patients. CONCLUSION: Three to seven years after NE, the patients had higher GFR and FF, more proteinuria, and higher ambulatory systolic BP compared with the healthy controls. NE may thus cause mild renal lesions and alterations in BP in some patients.

Bone isoenzyme of serum alkaline phosphatase and serum inorganic phosphate in metabolic bone disease of prematurity.

UNLABELLED: We wanted to improve detection of low bone mineral density in preterm infants by combining serum measurements of total alkaline phosphatase, its bone-type isoenzyme and serum inorganic phosphate in a prospective design. The subjects were 43 preterm infants. Total and bone isoenzyme activity of alkaline phosphatase was determined at 3 wk chronological age and at 3 and 6 mo corrected age. The main outcome measure, apparent bone mineral density (BMAD) at the distal forearm and forearm shaft, was assessed by dual energy X-ray absorptiometry at 3 and 6 mo corrected age. An apparent density below 95 mg/cm3 at 3 mo corrected age was considered to indicate bone disease, based on the distribution of BMAD values of children with non-complicated courses of prematurity. At 3 mo corrected age, total alkaline phosphatase activities exceeding 900 IU/l revealed low bone mineral density with 88% sensitivity and 71% specificity. Measurements of bone isoenzyme activity did not improve diagnostic performance. Serum inorganic phosphate levels below 1.8 mmol/l reflected low bone density with high specificity (96%), but the sensitivity was only 50%. CONCLUSION: A combination of the criteria "serum total alkaline phosphatase activity above 900 IU/l" and "serum inorganic phosphate concentrations below 1.8 mmol/l" yielded a sensitivity of 100% at a specificity of 70%. This was the best available screening method for low bone mineral density in preterms.

External quality assessment of urine analysis in Europe. Results of a round table discussion during the symposium 'From uroscopy to molecular analysis', Seeon, Germany, September 18-20, 1999.

Interlaboratory surveys on urine quantities have only recently been introduced in several European countries. Representatives of 10 European countries exchanged their experiences during an international urinalysis meeting held in September 1999. Although still not mandatory in most areas, more than 5000 laboratories participated in external quality assessment programs in the countries represented. Qualitative (test strips and microscopic morphology) as well as quantitative chemical and immunochemical quantities were included. The maximal allowable deviations are reported as well as methods used to determine target values. Consensus scales up to reference methods were applied. The participants agreed that quality criteria need to be defined separate from those already existing for plasma/serum analytes. Besides higher biological variables and different medical needs, less standardisation of methods to quantify urine constituents was observed as a major cause of higher interlaboratory differences.

Towards European urinalysis guidelines. Introduction of a project under European Confederation of Laboratory Medicine.

Improved standardized performance is needed because urinalysis continues to be one of the most frequently requested laboratory tests. Since 1997, the European Confederation of Laboratory Medicine (ECLM) has been supporting an interdisciplinary project aiming to produce European urinalysis guidelines. More than seventy clinical chemists, microbiologists and ward-based clinicians, as well as representatives of manufacturers are taking part. These guidelines aim to improve the quality and consistency of chemical urinalysis, particle counting and bacterial culture by suggesting optimal investigative processes that could be applied in Europe. The approach is based on medical needs for urinalysis. The importance of the pre-analytical stage for total quality is stressed by detailed illustrative advice for specimen collection. Attention is also given to emerging automated technology. For cost containment reasons, both optimum (ideal) procedures and minimum analytical approaches are suggested. Since urinalysis mostly lacks genuine reference methods (primary reference measurement procedures; Level 4), a novel classification of the methods is proposed: comparison measurement procedures (Level 3), quantitative routine procedures (Level 2), and ordinal scale examinations (Level 1). Stepwise strategies are suggested to save costs, applying different rules for general and specific patient populations. New analytical quality specifications have been created. After a consultation period, the final written text will be published in full as a separate document.

Evaluation of plasma cystatin C as a marker for glomerular filtration rate in patients with type 2 diabetes.

AIM: To evaluate plasma cystatin C as a marker of the glomerular filtration rate in patients with type 2 diabetes and their age and sex-matched controls. MATERIALS AND METHODS: Forty-seven patients with one decade of type 2 diabetes and 51 non-diabetic control subjects were studied. Plasma cystatin C was measured by particle-enhanced turbidimetric immunoassay in a new application for the Hitachi 704 analyzer. For comparison, plasma creatinine and creatinine clearance were measured. The plasma clearance of 51Cr-EDTA by the single injection method was utilized as reference. RESULTS: In patients with type 2 diabetes the correlation coefficient between plasma cystatin C and the plasma clearance of 51Cr-EDTA was 0.774 (Spearman's coefficient) and that between plasma creatinine and the plasma clearance of 51Cr-EDTA was 0.556 (p = 0.001 for the difference). The correlation between creatinine clearance and the plasma clearance of 51Cr-EDTA was 0.411. In receiver operating characteristic (ROC) curve analysis the diagnostic accuracy of plasma cystatin C was significantly better than that of plasma creatinine (p = 0.047) or creatinine clearance (p = 0.001). The best diagnostic efficiency (98%) for cystatin C was obtained when the cut-off limit was set at 1.32 mg/l. In the control group the correlation coefficients were: between cystatin C and the plasma clearance of 51Cr-EDTA 0.627, between creatinine and the plasma clearance of 51Cr-EDTA 0.466 and between creatinine clearance and the plasma clearance of 51Cr-EDTA 0.416. The area under the ROC plot curve of cystatin C was also greatest in the control group, but the diagnostic accuracy of cystatin C was marginally better than that of either plasma creatinine (p = 0.05) or creatinine clearance (p = 0.08). Among the control subjects various non-renal causes may have interfered with cystatin C concentrations reducing the correlations. CONCLUSIONS: Cystatin C measurement is a more sensitive and specific test for GFR in patients with type 2 diabetes than plasma creatinine or its clearance, when GFR is normal or only slightly reduced. If an elevated cystatin C concentration is found, non-renal factors have to be excluded. The turbidimetric application described here can easily be applied for most clinical chemistry analyzers and is therefore useful in daily clinical practice.

The occurrence of renal involvement in primary Sjögren's syndrome: a study of 78 patients.

OBJECTIVE: To ascertain the occurrence of renal involvement in patients with primary Sjögren's syndrome (pSS). METHODS: Urinary total protein excretion from 24 h urine collection, as well as urinary excretion rates of albumin, alpha-1 microglobulin (alpha1m) and IgG from overnight 8 h collections, were determined from 78 pSS patients (75 females, three males). Urine acidification capacity after oral ammonium chloride load was tested in 55 of these patients. RESULTS: Mild proteinuria (0.15-0.42 g/24 h) was observed in 34 patients (44%). Increased urinary excretion rates of albumin (>/=20 microgram/min), alpha1m (>/=7.0 microgram/min) or IgG (>/=5.0 microgram/min) were detected in nine (12%), nine (12%) and 11 patients (14%), respectively. Latent or overt distal renal tubular acidosis (dRTA) was observed in 18 out of 55 patients with pSS (33%). These patients had a longer duration of the disease (10+/-4 vs 8+/-4 yr; P

Evaluation of Sysmex UF-100 urine flow cytometer vs chamber counting of supravitally stained specimens and conventional bacterial cultures.

We evaluated the Sysmex UF-100 urine flow cytometer (TOA Medical Electronics, Kobe, Japan) with 269 uncentrifuged urine specimens by comparing it with Sternheimer staining and particle counting in 1-microL disposable chambers with both brightfield and phase-contrast microscopy (the reference method). Results of routine test strip analysis, sediment microscopy (182 specimens), and bacterial culture (204 specimens) were also available. Detection of urinary WBCs and RBCs was highly reliable with the UF-100 compared with manual chamber counting (r = .98 and .88, respectively). Identification of bacteria was equal to that with visual microscopy of uncentrifuged specimens; sensitivity was 55%, and specificity 90%, compared with bacterial cultures at a cutoff of > 10(3) colony-forming units per milliliter. Renal damage was difficult to evaluate even with manual methods because of the low counts of renal tubular cells and casts; with standard manual Sternheimer-stained sediment analysis, sensitivity was 65% to 69% and specificity 66% to 91%, compared with the uncentrifuged chamber method at a cutoff of 3 and 10 particles per microliter, respectively. Renal damage was demonstrated with the UF-100 with a sensitivity of 26% to 69% and specificity 92% to 94%, compared with chamber counts. Automated urinalysis with the UF-100 urine flow cytometer offers considerable savings in time and labor. When high sensitivity is needed, visual microscopic review should be performed to detect renal disease.

Randomised controlled trial of vitamin D supplementation on bone density and biochemical indices in preterm infants.

AIMS: To test the hypothesis that a vitamin D dose of 200 IU/kg, maximum 400 IU/day, given to preterm infants will maintain normal vitamin D status and will result in as high a bone mineral density as that attained with the recommended dose of 960 IU/day. METHODS: Thirty nine infants of fewer than 33 weeks of gestational age were randomly allocated to receive vitamin D 200 IU/kg of body weight/day up to a maximum of 400 IU/day or 960 IU/day until 3 months old. Vitamin D metabolites, bone mineral content and density were determined by dual energy x-ray absorptiometry, and plasma ionised calcium, plasma alkaline phosphatase, and intact parahormone measurements were used to evaluate outcomes. RESULTS: The 25 hydroxy vitamin D concentrations tended to be higher in infants receiving 960 IU/day, but the differences did not reach significance at any age. There was no difference between the infants receiving low or high vitamin D dose in bone mineral content nor in bone mineral density at 3 and 6 months corrected age, even after taking potential risk factors into account. CONCLUSIONS: A vitamin D dose of 200 IU/kg of body weight/day up to a maximum of 400 IU/day maintains normal vitamin D status and as good a bone mineral accretion as the previously recommended higher dose of 960 IU/day. Vitamin D is a potent hormone which affects organs other than bone and should not be given in excess to preterm infants.

Quality specifications for ordinal scale measurements with multiproperty (multiple) urine test strips.

Analytical quality specifications for ordinal scale measurements have not been presented so far. Criteria are suggested for multiproperty (multiple) urine test strips based on upper limits of healthy reference intervals, analytical performance and statistical tests applicable to ordinal scales. Trueness (accuracy) can be evaluated against an acceptable comparison method by applying sensitivity and specificity concepts, and defining a grey zone with a lower detection limit and an upper confirmation limit. Concordance (agreement) of two or more ordinal scale categories should be evaluated by subtracting random agreement, using Kappa statistics. Repeatability (precision) can be calculated for categorized results using binomial statistics.

Immune responses and clinical outcome of massive human osteoarticular allografts.

Cell mediated immune responses as measured by lymphocyte proliferation induced by the mitogens phytohemagglutinin and concanavalin A and antigen extracts of donor derived bone were studied within 2 years after wide resection of bone tumors in 18 patients receiving fresh frozen massive osteoarticular allografts. No uniform changes were seen in mitogen induced responses in 18 patients. However, five of nine patients tested with antigen extracts of donor derived bone showed elevated immune responses, one moderate and four weak. The incorporation of the allograft (evaluated by repeated radiographs; specific isotope techniques; clinical outcome assessed by the functional rating scores of Mankin-Waber and the Musculoskeletal Tumor Society; and histologic biopsy findings on new bone formation) did not differ in these patients from those in patients without any response to donor derived tissue. During a long term followup (mean, 11 years; range, 2-20 years), degenerative joint and sclerotic density bone changes developed after 2 to 4 years without correlation to immune responses. Histologic specimens showed no signs of immunologic reaction, and no clinical rejection episodes were recorded. A slightly variable immune response to allograft bone seems to occur, but its clinical significance for outcome of the grafts remains to be determined. The low immune responses might reflect a low antigen release rate through an indirect pathway or immunologic tolerance to antigens or proteins shed from massive allografts that are nonliving scaffold implants during the creeping substitution process, corresponding to the low immune response and the slow histologic repair.

Multicentre evaluation of the urine analyser Miditron Junior.

A multicentre evaluation of the urine analyser Miditron Junior was performed at four laboratories. The Miditron Junior analyser provides semi-quantitative results for erythrocytes, bilirubin, urobilinogen, ketone bodies, glucose, protein, nitrite, leukocytes, pH and relative density. Accuracy of the Miditron Junior analyser was evaluated by comparison to quantitative analytical chemistry methods (glucose, total protein), physical methods (pH, relative density), and microscopic methods (erythrocytes, leukocytes). Agreement was defined as identical or neighbouring concentration block. The level of agreement found with chemical, physical or microscopic methods for the six analytes tested varied from 79 to 99%. The within-run precision was determined as repeatability by using 31 native urines. The results of repeated measurements (n = 10) fell in the same concentration block, or in case of borderline concentration were spaced between two adjacent colour blocks. Day-to-day precision covering a minimum of 20 days using commercially available control solutions yielded results within +/- one colour block from the mean.

Estimation of reference change limits using patient data.

Two approaches for deriving reference change limits from patient data are described. In the direct method, hospital database information is used for the selection of appropriate reference groups. If database information is not sufficient or reliable enough, but still most of the source data can be considered as health-related, an indirect method can be applied in the calculation of rough estimates for reference change limits. A computer program developed by us, GraphROC for Windows, includes both methods for the estimation of change limits from patient data. Time between specimen collections should be included as one classifying factor in the selection of source data. When only one previous result is available for comparison, change limits based on the reference sample group form the only available guide for clinical interpretation. However, when several previous results are available and the within-subject variances for the considered analyte are known to be heterogeneous between individuals, the clinical interpretation should rather be based on application of time series analysis.

Reliability and adequacy of discharge diagnosis databases in the production of reference values.

Discharge diagnoses provide a possibility to select patients individually and then to establish reference values for both "pathological" and control groups. Currently, the available diagnostic information is still at its infancy and should be carefully evaluated before the reference values based on those groups are utilized. It is anticipated that electronic storage of diagnostic and therapeutic information will be applied more commonly in the future as the development of computers makes it easier. The advanced utilization of laboratory data challenges physicians both in the clinical and laboratory side to participate in this development in order to make the information systems serve their actual needs more closely.