Walter Hofmann

Physiopathology of Proteinuria and Laboratory Diagnostic Strategy Based on Single Protein Analysis

Abstract A quantification of proteins of different molecular size has been shown to be useful in characterizing the mechanism and medical causes of proteinuria. By analyzing urine albumin, α1-microglobulin, immunoglobulin G and α2-macroglobulin together with total protein, prerenal, glomerular, tubular and postrenal causes of proteinuria can be detected and differentiated by their specific urine protein patterns. Using automated turbidimetric procedures, prerenal proteinurias are characterized by an albumin/total protein ratio below 0.4. Tubulo-interstitial diseases which are negative in the protein test strip procedure are detected and clearly differentiated from other causes of proteinuria by their high α1-microglobulin/albumin ratios. In postrenal proteinuria, α2-macroglobulin proved to be a useful marker, when albumin excretion exceeds 100 mg/l urine. This protein exhibits plasma-like ratios to albumin in postrenal causes, whereas it is much lower in renal proteinurias. The new strategy, which has been evaluated in more than 500 clinically and partly histologically proven cases of renal diseases, more sensitively detects glomerular and tubulo-interstitial diseases when applied in urine screening and allows us to distinguish all clinically important causes from analysis of a morning spot urine sample.

Clinical role of urinary low molecular weight proteins: their diagnostic and prognostic implications.

In traditional urinalysis, casts in the urinary sediment are the only specific signs of renal tubular injury. When tubulo‐interstitial fibrosis became the most predictive sign of renal outcome, tubular enzymes derived from proximal tubular brush border or lysosomes were used as early markers of nephrotoxicity and other tubular dysfunctions. More recently, the increase in low molecular weight proteins in urine, assumed to be freely filtered, was reported to reflect tubular dysfunction. This can have pre‐renal, renal and post‐renal causes. Among the pre‐renal causes, Bence Jones protein (immunoglobulin light chains), myoglobin and haemoglobin are signs of extra‐renal diseases. On the other hand, β2‐microglobulin, α1‐microglobulin, retinol binding protein and lysozyme were recommended as tubular markers. Because of its lower pre‐renal variability and higher stability in urine during storage in the bladder and urinary vessel, α1‐microglobulin proved to be the most valuable in early detection, renal outcome prediction and easy inclusion in routine analytical programmes. In addition, other markers of intra‐renal inflammatory processes may help to mirror histological changes occurring in the kidney. Future guidelines should therefore include low molecular protein as a tubular marker.

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.

NGAL, L-FABP, and KIM-1 in comparison to established markers of renal dysfunction

Abstract Background: New urinary biomarkers like neutrophil gelatinase-associated lipocalin (NGAL), liver-type fatty acid binding protein (L-FABP), and kidney injury molecule-1 (KIM-1) open the opportunity to detect kidney injuries in early stages. Our study aimed at evaluating NGAL, L-FABP, and KIM-1 in comparison to established markers of urine protein differentiation for detection of renal dysfunction. Methods: On the basis of the PROTIS expert system (for differentiation of glomerulo-/tubulopathy) urine and plasma samples of 263 randomly selected patients were routinely examined (urine: total protein, albumin, IgG, α1-microglobulin, creatinine, and dip stick results for leukocytes, blood, protein, glucose, pH, and nitrite; plasma: creatinine and cystatin C) followed by the analysis of the new urine biomarkers NGAL (CMIA), L-FABP (ECLIA), and KIM-1 (ELISA). Results: Of the three new markers L-FABP showed the highest correlation with α1-microglobulin (r=0.76, p<0.01) and was closest associated with the degree of tubular proteinuria assessed by the PROTIS system. NGAL distinguished the PROTIS proteinuria groups with distinctive tubular proteinurias from the controls as well, but revealed a marked diagnostic influence by leukocyturia. Urinary KIM-1 revealed only a weak diagnostic value for the detection of renal injury. Conclusions: Urinary NGAL and L-FABP proved to be promising candidates for detecting injuries of the renal tubular system over a broad range of clinical conditions. L-FABP showed a better diagnostic performance and a lower interference by leukocyturia and hematuria than NGAL. Both markers may serve as sensitive tissue injury markers in addition to the established markers of renal functional impairment.

Human neutral brush border endopeptidase EC 3.4.24.11 in urine, its isolation, characterisation and activity in renal diseases

Human neutral brush border endopeptidase (NEP) was purified from the urine of patients suffering from acute toxic tubulointerstitial nephropathy. An enzyme preparation with specific activity of 102 Ug(-1) protein was obtained. The urinary activities of neutral endopeptidase and alanine aminopeptidase were measured in patients with renal disease and in 30 control patients, resulting in a reference range from 0.1 to 0.7 Ug(-1) creatinine and 1.4-14.1 Ug(-1) creatinine, respectively. Urine enzyme activities were highest in patients with acute tubulotoxic renal diseases. Neutral endopeptidase and alanine aminopeptidase activities were found to be 6.5- and 10-fold higher than the upper value of the reference range, respectively. Smaller increases in the rate of excretion of these enzymes (2.5- and 3.5-fold), respectively, were observed in patients suffering from acute tubular insufficiency and even lower increases, 2- and 1.5-fold, respectively, were observed in patients with chronic renal diseases. In diabetics and kidney transplant patients the enzyme excretion rates were within the reference range. Assay of both transmembrane metalloproteinases in urine may prove valuable in serving as markers for renal toxicity. Together with beta-NAG these enzymes could be employed as differentiation markers between acute and chronic tubular insufficiency.

Benefits and limitations of laboratory diagnostic pathways

Abstract Diagnostic pathways are an essential subset of clinical pathways and a logical consequence of DRG-based reimbursement. They combine the principle of stepwise reflex and reflective testing with a management concept that helps to fulfill medical needs with organizational and economic efficacy. The two most common formats describing diagnostic pathways are graphical decision trees on paper and “if…then…else” rules on computers. From a laboratory point of view, diagnostic pathways represent “smart” test profiles, which – in contrast to conventional (inflexible) profiles – are not necessarily worked off completely, but just to a point, where a diagnostic decision can be made. This improves the cost-effectiveness of laboratory testing, while making sure that no essential tests are missed. The paper describes benefits and limitations of diagnostic pathways from a medical, organizational, and economic point of view. Their major advantage is also their major drawback, since they make the diagnostic process on the one hand extremely straight-forward and transparent, while on the other hand oversimplifying the underlying medical decision principles. This may provoke the abuse of their primarily medical intentions for mere economic purposes.

Multicentre evaluation of KONE Optima analysis system

Abstract Optima from KONE Instruments Corporation is a new selective laboratory analyzer for turbidimetric, colorimetric and ion selective electrode measurements. Overall analytical performance of Optima and reagents provided by KONE was evaluated according to ECCLS guidelines, in a multicentre study involving four different laboratories, including substrates (cholesterol, high-density lipoprotein-cholesterol, creatinine), specific proteins (transferrin, IgG), enzyme activities (γ-glutamyltransferase, alanine aminotransferase) and electrolytes (sodium, potassium, chloride). The results obtained attest good precision of the system for all the analytes tested: the range of withinrun CV is 0.5%–4.3%, and range of between-day CV% is 0.8%–7.9% (median of four laboratories). Except for total cholesterol (5 % overestimation compared to the reference method) accuracy of measurement is adequate. Creatinine and uric acid assays were subjects of interference (defined as deviation > 10% from target value) by bilirubin and haemoglobin respectively.

Diagnostische Pfade bei Nierenerkrankungen 1) / Diagnostic pathways for renal diseases

Zusammenfassung Die Deutsche Vereinte Gesellschaft für Klinische Chemie und Laboratoriumsmedizin hat 2006 eine Arbeitsgruppe aus der Taufe gehoben, die gemeinsam mit Mitgliedern der Deutschen Gesellschaft für Nephrologie die bisher verfügbaren Empfehlungen zum Ausschluss, zur Erfassung und Differenzierung von Nierenerkrankungen als diagnostische Pfade strukturiert niederlegen. Die so geschaffene Grundlage soll dann mit EDV-Unterstützung als ein entsprechendes Konzept in der Klinik verfügbar bzw. anforderbar gemacht werden. Die vorliegende Arbeit enthält die Inhalte dieser Pfade betreffend die glomeruläre Filtrationsrate, Hämaturie, Leukozyturie und Proteinurie. Abstract In 2006 the German United Society for Clinical Chemistry and Laboratory Medicine together with the German Society of Nephrology founded a working group intending to develop diagnostic pathways for the exclusion, detection and differentiation of renal diseases. Based on existing recommendations these pathways may be structured to be a basis for implementation into hospital and laboratory information systems. The present paper describes the contents of these pathways regarding glomerular filtration rate, haematuria, leucocyturia and proteinuria.

PROTIS: Use of Combined Biomarkers for Providing Diagnostic Information on Disease States

We describe herein PROTIS, a software package that allows for the combination of urine biomarkers (proteins, small molecules, cells) to provide accurate diagnostic information on renal disease states. Such an approach exemplifies the advantage conferred by using multiple markers, and can be generalized for identification of biomarker signatures to separate distinct sample groups.