Lothar Siekmann

Creatinine Measurement: State of the Art in Accuracy and Interlaboratory Harmonization

CONTEXT The National Kidney Disease Education Program recommends calculating glomerular filtration rate from serum creatinine concentration. Accurate creatinine measurements are necessary for this calculation. OBJECTIVE To evaluate the state of the art in measuring serum creatinine, as well as the ability of a proficiency testing program to measure bias for individual laboratories and method peer groups. DESIGN A fresh-frozen, off-the-clot pooled serum specimen plus 4 conventional specimens were sent to participants in the College of American Pathologists Chemistry Survey for assay of creatinine. Creatinine concentrations were assigned by isotope dilution mass spectrometry reference measurement procedures. PARTICIPANTS Clinical laboratories with an acceptable result for all 5 survey specimens (n = 5624). RESULTS The fresh frozen serum (FFS) specimen had a creatinine concentration of 0.902 mg/dL (79.7 micromol/L). Mean bias for 50 instrument-method peer groups varied from -0.06 to 0.31 mg/dL (-5.3 to 27.4 micromol/L), with 30 (60%) of 50 peer groups having significant bias (P < .001). The bias variability was related to instrument manufacturer (P < or = .001) rather than method type (P = .02) with 24 (63%) of 38 alkaline picric acid methods and with 6 (50%) of 12 enzymatic methods having significant biases. Two conventional specimens had creatinine concentrations of 0.795 and 2.205 mg/dL (70.3 and 194.9 micromol/L) and had apparent survey biases significantly different (P < .001) from that of the FFS specimen for 34 (68%) and 35 (70%) of 50 peer groups, respectively. CONCLUSIONS Thirty of 50 peer groups had significant bias for creatinine. Bias was primarily associated with instrument manufacturer, not with type of method used. Proficiency testing using a commutable specimen measured participant bias versus a reference measurement procedure and provided trueness surveillance of instrument-method peer groups.

IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase.

Abstract This paper is the fourth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37°C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 6. Reference Procedure for the Measurement of Catalytic Concentration of γ-Glutamyltransferase; Part 7. Certification of Four Reference Materials for the Determination of Enzymatic Activity of γ-Glutamyltransferase, Lactate Dehydrogenase, Alanine Aminotransferase and Creatine Kinase at 37°C. A document describing the determination of preliminary upper reference limits is also in preparation. The procedure described here is deduced from the previously described 30°C IFCC reference method (1). Differences are tabulated and commented on in Appendix 2.

DETERMINATION OF STEROID HORMONES BY THE USE OF ISOTOPE DILUTION–MASS SPECTROMETRY: A DEFINITIVE METHOD IN CLINICAL CHEMISTRY

Isotope dilution-mass spectrometry (ID-MS) is a reliable analytical tool for the highly accurate measurement of volatile compounds in biological fluids. In the present investigation the technique of ID-MS has been applied to the specific and accurate determination of oestradiol-17β, oestriol, testosterone, progesterone, aldosterone and cortisol in human serum. The analytical procedure for the measurement of steroid hormones comprises the following steps: (1) addition of a 14C-labelled steroid to the serum sample; (2) extraction of the 14C-labelled and of the non-labelled steroid by the use of an organic solvent; (3) purification of the steroid fraction by column chromatography on Sephadex LH-20 or on Lipidex-5000; (4) formation of a derivative; (5) combined gas chromatography-mass spectrometry. During gas chromatography the mass spectrometer continuously records two m/e values corresponding to the labelled and the non-labelled hormone. Quantitative determination is performed by comparing the peak heights of the hormone to be determined and of the labelled internal standard. The accuracy of the methods is based on the high specificity of mass spectrometry and on the exact control of recovery employing the principle of isotope dilution. Therefore, the analytical procedures described here may be recommended as definitive methods in clinical chemistry.

IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37°C

Abstract This paper is the second in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37°C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 4. Reference Procedure for the Measurement of Catalytic Concentration of Alanine Aminotransferase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 6. Reference Procedure for the Measurement of Catalytic Concentration of γ-Glutamyltransferase; Part 7. Certification of Four Reference Materials for the Determination of Enzymatic Activity of γ-Glutamyltransferase, Lactate Dehydrogenase, Alanine Aminotransferase and Creatine Kinase at 37°C. A document describing the determination of preliminary reference values is also in preparation. The procedure described here is deduced from the previously described 30°C IFCC reference method (1). Differences are tabulated and commented on in Appendix 3.

Steroid sulfatase (STS) expression in the human temporal lobe: enzyme activity, mRNA expression and immunohistochemistry study.

Dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) are suggested to be important neurosteroids. We investigated steroid sulfatase (STS) in human temporal lobe biopsies in the context of possible cerebral DHEA(S) de novo biosynthesis. Formation of DHEA(S) in mature human brain tissue has not yet been studied. 17α‐Hydroxylase/C17‐20‐lyase and hydroxysteroid sulfotransferase catalyze the formation of DHEA from pregnenolone and the subsequent sulfoconjugation, respectively. Neither their mRNA nor activity were detected, indicating that DHEA(S) are not produced within the human temporal lobe. Conversely, strong activity and mRNA expression of DHEAS desulfating STS was found, twice as high in cerebral neocortex than in subcortical white matter. Cerebral STS resembled the characteristics of the known placental enzyme. Immunohistochemistry revealed STS in adult cortical neurons as well as in fetal and adult Cajal‐Retzius cells. Organic anion transporting proteins OATP‐A, ‐B, ‐D, and ‐E showed high mRNA expression levels with distinct patterns in cerebral neocortex and subcortical white matter. Although it is not clear whether they are expressed at the blood–brain barrier and facilitate an influx rather than an efflux, they might well be involved in the transport of steroid sulfates from the blood. Therefore, we hypothesize that DHEAS and/or other sulfated 3β‐hydroxysteroids might enter the human temporal lobe from the circulation where they would be readily converted via neuronal STS activity.

Impairment of Adrenocortical Function Associated with Increased Plasma Tumor Necrosis Factor-Alpha and Interleukin-6 Concentrations in African Trypanosomiasis

African sleeping sickness (SS) is a severe, potentially lethal parasitic disease. The treatments of choice are the antiparasitic agents suramin, which is adrenotoxic, and/or melarsoprol. We evaluated the functional integrity of the hypothalamic-pituitary-adrenal (HPA) axis of patients with SS before, during, and after therapy with suramin and/or melarsoprol, in two sequential stages. First, we employed the standard adrenocorticotropic hormone (ACTH) 1-24 stimulation test (250 micrograms i.v.) to assess the maximal adrenocortical responsiveness of 69 patients with SS and 38 normal controls. We demonstrated paradoxically subnormal cortisol responses before suramin therapy [net cortisol response 60 min after stimulation: 10.5 +/- 2.9 (mean +/- SE) vs. 17.5 +/- 1.0 micrograms/dl for controls, p = 0.004], with 27% of the patients falling within the adrenal insufficiency range (stimulated cortisol concentration < 20 micrograms/dl). These responses subsequently and unexpectedly improved with suramin and/or melarsoprol therapy. Second, we performed a human corticotropin-releasing hormone (hCRH) test (100 micrograms i.v.) in 68 additional patients with SS and 14 control subjects to examine whether the glucocorticoid deficiency observed was primary and/or secondary. Compared to controls, the ACTH and cortisol responses to hCRH were blunted (ACTH after 60 min: 29 +/- 7 vs. 58 +/- 8 pg/ml in controls, p = 0.014; cortisol: 15.2 +/- 1.5 vs. 19.6 +/- 0.7 micrograms/dl, p = 0.018), suggesting the presence of secondary adrenal insufficiency. There was improvement of both ACTH and cortisol responsiveness to hCRH with therapy, with cortisol recovery occurring before ACTH, suggesting an additional primary component of adrenal dysfunction in these patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the dehydroepiandrosterone (DHEA) metabolism via oxysterol 7α-hydroxylase and 17-ketosteroid reductase activity in the human brain

Dehydroepiandrosterone and its sulphate are important factors for vitality, development and functions of the CNS. They were found to be subjects to a series of enzyme‐mediated conversions within the rodent CNS. In the present study, we were able to demonstrate for the first time that membrane‐associated dehydroepiandrosterone 7α‐hydroxylase activity occurs within the human brain. The cytochrome P450 enzyme demonstrated a sharp pH optimum between 7.5 and 8.0 and a mean KM value of 5.4 µm, corresponding with the presence of the oxysterol 7α‐hydroxylase CYP7B1. Real‐time RT–PCR analysis verified high levels of CYP7B1 mRNA expression in the human CNS. The additionally observed conversion of dehydroepiandrosterone via cytosolic 17β‐hydroxysteroid dehydrogenase activity could be ascribed to the activity of an enzyme with a broad pH optimum and an undetectably high KM value. Subsequent experiments with cerebral neocortex and subcortical white matter specimens revealed that 7α‐hydroxylase activity is significantly higher in the cerebral neocortex than in the subcortical white matter (p < 0.0005), whereas in the subcortical white matter, 17β‐hydroxysteroid dehydrogenase activity is significantly higher than in the cerebral neocortex (p < 0.0005). No sex differences were observed. In conclusion, the high levels of CYP7B1 mRNA in brain tissue as well as in a variety of other tissues in combination with the ubiquitous presence of 7α‐hydroxylase activity in the human temporal lobe led us to assume a neuroprotective function of the enzyme such as regulation of the immune response or counteracting the deleterious effects of neurotoxic glucocorticoids, rather than a distinct brain specific function such as neurostimulation or neuromodulation.

State of the Art in Trueness and Interlaboratory Harmonization for 10 Analytes in General Clinical Chemistry

CONTEXT Harmonization and standardization of results among different clinical laboratories is necessary for clinical practice guidelines to be established. OBJECTIVE To evaluate the state of the art in measuring 10 routine chemistry analytes. DESIGN A specimen prepared as off-the-clot pooled sera and 4 conventionally prepared specimens were sent to participants in the College of American Pathologists Chemistry Survey. Analyte concentrations were assigned by reference measurement procedures. PARTICIPANTS Approximately 6000 clinical laboratories. RESULTS For glucose, iron, potassium, and uric acid, more than 87.5% of peer groups meet the desirable bias goals based on biologic variability criteria. The remaining 6 analytes had less than 52% of peer groups that met the desirable bias criteria. CONCLUSIONS Routine measurement procedures for some analytes had acceptable traceability to reference systems. Conventionally prepared proficiency testing specimens were not adequately commutable with a fresh frozen specimen to be used to evaluate trueness of methods compared with a reference measurement procedure.

Establishing a reference system in clinical enzymology.

Abstract The goal of standardization for measurements of catalytic concentrations of enzymes is to achieve comparable results in human samples, independent of the reagent kits, instruments and laboratory where the procedure is carried out. To pursue this objective, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has launched a project to establish a reference system in clinical enzymology. This system is based on three hinges: a) extensively evaluated and carefully described reference procedures, b) certified reference materials and c) a network of reference laboratories operating in a highly controlled manner. The original IFCC-recommended procedures for alanine aminotransferase, aspartate aminotransferase, creatine kinase, γ-glutamyltransferase, lactate dehydrogenase and α-amylase have been slightly modified to optimize them at 37 °C, with the definition of detailed operating procedures. A group of laboratories perform these procedures manually, with selfmade reagents on carefully calibrated instruments. Partially purified and stabilized materials, prepared in the past by the Community Bureau of Reference, have been re-certified by these laboratories for alanine aminotransferase, creatine kinase, γ-glutamyltransferase and lactate dehydrogenase activities. Using these materials and the manufacturers standing procedures, industry can assign traceable values to commercial calibrators. Thus, clinical laboratories, which will use routine procedures with these validated calibrators to measure human specimens, can finally obtain values which are traceable to reference procedures.

IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C.

Abstract This paper is the eighth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37°C and the certification of reference preparations. Other parts deal with: Part 1. The concept of reference procedures for the measurement of catalytic activity concentrations of enzymes; Part 2. Reference procedure for the measurement of catalytic concentration of creatine kinase; Part 3. Reference procedure for the measurement of catalytic concentration of lactate dehydrogenase; Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase Part 6. Reference procedure for the measurement of catalytic concentration of γ-glutamyltransferase; Part 7. Certification of four reference materials for the determination of enzymatic activity of γ-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase at 37°C. The procedure described here is deduced from the previously described 30°C IFCC reference method. Differences are tabulated and commented on. Clin Chem Lab Med 2006;44:1146–55.This paper is the eighth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C and the certification of reference preparations. Other parts deal with: Part 1. The concept of reference procedures for the measurement of catalytic activity concentrations of enzymes; Part 2. Reference procedure for the measurement of catalytic concentration of creatine kinase; Part 3. Reference procedure for the measurement of catalytic concentration of lactate dehydrogenase; Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase Part 6. Reference procedure for the measurement of catalytic concentration of gamma-glutamyltransferase; Part 7. Certification of four reference materials for the determination of enzymatic activity of gamma-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase at 37 degrees C. The procedure described here is deduced from the previously described 30 degrees C IFCC reference method. Differences are tabulated and commented on.