Preparation, stability and commutability of candidate reference materials for lactate dehydrogenase (LDH).

BACKGROUND: Lactate dehydrogenase (LDH) is a key enzyme that functions as a marker of cell damage. Its activity can be measured by a variety of laboratory methods. To eliminate inter-method bias and enhance equivalence among different measurement procedures, LDH detection needs to be standardized. METHODS: Optimized sequences coding for lactate dehydrogenase subunit A (LDH-A) and subunit B (LDH-B) were synthesized and cloned into the pRSFDuet vector, and then the constructed 6His-LDHA-pRSFDuet, 6His-LDHB-pRSFDuet, and 6His-LDHA-LDHB-pRSFDuet plasmids were transformed into Escherichia coli BL21 (DE3) for expression. The enzyme activity and specific activity of recombinant LDHs were detected. Electrophoresis of LDH isoenzymes was performed. The stability of recombinant LDHs and serum LDH was evaluated. Commutability of recombinant LDH-B was studied by the IFCC reference method and six routine methods. RESULTS: Three plasmids were constructed and three highly concentrated recombinant LDH isoenzymes were obtained. The specific activities of LDH-A, LDH-AB, and LDH-B were 18.08 U/mg, 21.74 U/mg, and 14.18 U/mg, respectively. There was a desirable linear correlation between the activities of recombinant LDH isoenzymes and their protein concentrations. Electrophoresis of LDH isoenzymes showed that the recombinant LDH-B corresponded to LDH1 and it demonstrated good stability at 4 °C and 25 °C for 5 weeks. LDH-B formulations in saline-bovine serum albumin solution and human serum matrix were commutable for six routine methods. CONCLUSION: Human recombinant LDH-B has great potential to become an improved and less expensive standard or reference material in external quality assessment for clinical LDH measurement.

Underfilling of vacuum blood collection tubes leads to increased lactate dehydrogenase activity in serum and heparin plasma samples.

Background Lactate dehydrogenase (LD) activity is routinely monitored for therapeutic risk stratification of malignant diseases, but is also prone to preanalytical influences. Methods We systematically analyzed the impact of defined preanalytical conditions on the hemolysis-susceptible parameters LD, potassium (K) and hemolysis index in vacuum blood collection tubes (serum [SE], heparin plasma [HP]). Blood was collected by venipuncture from healthy volunteers. Tubes were either filled or underfilled to approximately 50%, then processed directly or stored at room temperature for 4 h. Potassium (K), sodium (Na), chloride (Cl), LD, creatine kinase (CK), total cholesterol, and indices for hemolysis, icterus, and lipemia were analyzed. Filling velocity was determined in a subset of tubes. Findings in healthy volunteers were reconfirmed in an in-patient cohort (n = 74,751) that was analyzed for plasma yield and LD data distribution. Results LD activity was higher in HP compared to SE. Underfilling led to higher LD values (SE: +21.6%; HP: +28.3%), K (SE: +4.2%; HP: +5.3%), and hemolysis index (SE: +260.8%; HP: +210.0%), while other analytes remained largely unchanged. Filling velocity of tubes was approximately 3-fold higher in the first half compared to the second half in both HP and SE collection tubes. Importantly, plasma yield also inversely correlated with LD in routine patients. By calculating reference limits, the lowest plasma yield quartile of the patient cohort displayed LD values clearly exceeding current reference recommendations. Conclusions Underfilling of tubes leads to a higher proportion of blood aspirated with high velocity and relevant elevations in LD. This finding should be considered in cases of clinically implausible elevated LD activities.

The use of Fionet technology for external quality control of malaria rapid diagnostic tests and monitoring health workers' performance in rural military health facilities in Tanzania.

INTRODUCTION: Internal and external quality control (QC) of rapid diagnostic tests (RDTs) is important to increase reliability of RDTs currently used to diagnose malaria. However, cross-checking of used RDTs as part of quality assurance can rarely be done by off-site personnel because there is no guarantee of retaining visible test lines after manufacturers' recommended reading time. Therefore, this study examined the potential of using Fionet™ technology for remote RDT quality monitoring at seven clinics, identifying reasons for making RDT processing and interpretation errors, and taking corrective actions for improvement of diagnosis and consequently improved management of febrile patients. METHODS: The study was conducted at seven military health facilities in Mainland Tanzania and utilized RDTs capable of detecting Plasmodium falciparum specific Histidine-rich protein 2 (Pf-HRP2) and the genus specific Plasmodium lactate dehydrogenase (pLDH) for other species of plasmodium (P. vivax, P. malariae or P. ovale; pan-pLDH). Patients' data and images of processed RDTs from seven clinics were uploaded on a Fionet web portal and reviewed regularly to monitor preparation procedures and visual interpretation of test results compared to automated analysis using the Deki reader of RDT. Problems detected were rapidly communicated to remote laboratory personnel at the clinic for corrective action and follow-up of patients who were falsely diagnosed as negative and missed treatment. Factors contributing to making errors in visual interpretation of RDT results were analyzed during visits to the health facilities. RESULTS: A total of 1,367 (1.6%) out of 83,294 RDT test images uploaded to the Fionet portal had discordant test results of which 822 (60.1%) and 545 (39.9%) were falsely reported as negative and positive, respectively. False negative and false positive test results were common for a single test line in 515 (62.7%) and 741 (54.2%) tests, respectively. Out of 1,367 RDT images assessed, 98 (7.2%) had quality problems related to preparation procedures of which 95(96.9%) errors were due to putting too much blood on the sample well or insufficient buffer in the respective wells. The reasons for discrepant results included, false reporting of none existent lines in 526 (38.5%) tests, missing a faint positive line in 493 (36.1%), missing a strong positive line in 248(18.1%) and errors caused by poorly processed RDTs in 96 (7.2%) tests. Among the false negative tests (n = 822), 669 (48.9%) patients were eligible for follow-up and only 339 (48.5%) were reached and 291 (85.8%) received appropriate anti-malaria therapy. CONCLUSION: Fionet technology enabled remote monitoring of RDT quality issues, identifying reasons contributing to laboratory personnel making errors and provided a rapid method to implement corrective actions at remote sites to improve malaria diagnosis and consequently improved health care management of febrile patients infected with malaria.

Verification study on the NORIP LDH reference intervals with a proposed new upper reference limit.

We verified the lactate dehydrogenase (LDH) reference interval (RI) provided by the Nordic Reference Interval Project (NORIP). The serum LDH concentration was analysed on the Dimension Vista 1500 system with an IFCC method with a bias of +2.1 % and +2.7 % against NFKK Reference Serum X and ERM-AD453/IFCC, respectively, showing verification of transference of the NORIP RI. Selective data mining in clinical laboratory information systems for retrospective serum LDH test results was used to calculate an indirect RI. For the adult age group (18 to <70 years) the limits of the interval was 127 U/L (90 % CI: 123-132 U/L) and 240 U/L (90 % CI: 234-243 U/L). However, the NORIP upper limit for the adult age group is 205 U/L (90 % CI: 198-210 U/L). Accordingly, 25.1 % of LDH test results were above the NORIPs upper limit of 205 U/L. If LDH analysis was requested by the hospital's medical departments, outpatient clinics or general practitioners 29.2 %, 26.2 % and 20.9 %, respectively, were above the 205 U/L limit. Differences in transport time before centrifugation of blood, and different transport principles could not explain the relative high percent of test results above the NORIP 205 U/L limit. The indirect finding of an upper limit of 240 U/L (90 % CI: 234-243 U/L), and the relative high number of test result >205 U/L, suggests that the NORIP upper limit should be adjusted.

Reconsidering the H&E stain as the gold standard in assessing the depth of burn wounds.

While histological examination is considered by most as the gold standard for burn depth assessment, it has no practical use in the clinical setting. It has, however, been used in the research setting, as a mean for evaluating emerging techniques of depth measurement. Due to the limitations of the H&E stain, other stains have also been explored, such as lactate dehydrogenase (LDH), as presented in this issue, in "Improving the Histologic Characterization of Burn Depth." As the determination of burn depth is not a typical subject in dermatopathology, a summary of selected techniques and the possible role for the LDH stain in future research, is described herein.

Metrological traceability of values for catalytic concentration of enzymes assigned to a calibration material.

BACKGROUND: The metrological traceability of values for the catalytic concentration of several enzymes assigned to a calibration material has been assured by following the recently published International Standard ISO 18153. METHODS: A traceable value with a measurement uncertainty was assigned for the catalytic concentration of alanine aminotransferase, creatine kinase, gamma-glutamyltransferase and lactate dehydrogenase in two materials from different sources. These are all measurable quantities, with the primary reference measurement procedure described by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and a primary calibrator giving metrological traceability to the SI unit of measurement. The metrologically traceable calibration was validated by measuring human serum samples using the primary reference measurement procedure and a routine commercial measurement procedure calibrated with the traceable materials. RESULTS: Results showed that the primary reference procedure, selected manufacturers' procedures and the end-user's routine procedure for each enzyme have the same analytical specificity. Four of eight commercial calibrators tested were commutable, whereas the others had a very small difference in absolute terms, indicating that these materials would be useful for calibration. CONCLUSION: The implementation of a reference system for enzyme measurements was demonstrated that assures the traceability of patient results to SI units.

New IFCC reference procedures for the determination of catalytic activity concentrations of five enzymes in serum: preliminary upper reference limits obtained in hospitalized subjects.

Consensus among clinical chemists has dictated a change in reference temperature for enzyme catalytic concentrations from 30 to 37 degrees C. Consequently, International Federation of Clinical Chemistry (IFCC) reference procedures have been redefined at the latter temperature. Acceptance in practice of these new procedures requires well-established reference values and clinical decision limits, but the establishment of reference values is complex. Therefore, as a provisional approach and to facilitate early application of the new IFCC procedures, we report our experience gained with them in the transfer of values from the consensus methods used hitherto in Germany to the new procedures. The preliminary upper reference limits were determined for catalytic activity concentrations of the enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), gamma-glutamyltransferase (gamma-GT) and lactate dehydrogenase (LDH) in human sera. Since enzyme measurements are almost always made on sera from non-ambulant subjects, we have used hospital patients aged 17 years and older as the subjects of our study. The catalytic activity concentrations obtained by measurements with the German consensus methods for the respective enzyme were chosen in combination with additional enzymes of similar diagnostic relevance to classify patients' samples as part of the respective reference collective. Measurements for the determination of the upper reference limits were performed manually by use of the primary reference procedures at the measurement temperature 37 degrees C according to IFCC, and also by employing mechanized measurements adapted to the reference procedures. The upper reference limits were calculated as the 97.5th percentile of the reference collectives and determined separately for women and men: ALT: 34 U/l (female) and 45 U/l (male); AST: 31 U/l (female) and 35 U/l (male); CK: 145 U/l (female) and 171 U/l (male); gamma-GT: 38 U/l (female) and 55 U/l (male); LDH: 247 U/l (female) and 248 U/l (male).

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 7. Certification of four reference materials for the determination of enzymatic activity of gamma-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase accord.

This paper is the seventh 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. A document describing the determination of preliminary reference values is also in preparation. The certification of the catalytic activity concentrations as determined by the recently elaborated IFCC primary reference methods at 37 degrees C of four enzyme preparations, namely IRMM/IFCC 452 (gamma-glutamyltransferase), IRMM/IFCC 453 (lactate dehydrogenase 1), IRMM/IFCC 454 (alanine aminotransferase) and IRMM/IFCC 455 (creatine kinase) is described. Homogeneity data were derived from previous results. Stability was assessed using recently obtained data as well as data from previous stability studies. The collaborative study for value assignment was performed under a strict quality control scheme to ensure traceability to the primary reference method. Uncertainty of the materials was assessed in compliance with the Guide to the Expression of Uncertainty in Measurement. The certified values obtained at 37 degrees C are 1.90 microkat/l +/- 0.04 microkat/l (114.1 U/l +/- 2.4 U/l), for gamma-glutamyltransferase, 8.37 microkat/l +/- 0.12 microkat/l (502 U/l +/- 7 U/l), for lactate dehydrogenase 1, 3.09 microkat/l +/- 0.07 microkat/l (186 U/l +/- 4 U/l), for alanine aminotransferase and 1.68 microkat/l +/- 0.07 microkat/l (101 U/l +/- 4 U/l), for creatine kinase. The materials are intended for internal quality control as well as for the evaluation of test systems as required by recent European Union legislation. Furthermore, the materials can be used to transfer accuracy from a reference method to a routine procedure provided the procedures exhibit the same analytical specificity and the certified materials are commutable.

[Elevation of serum lactate dehydrogenase. Diagnostic, prognostic and evolutive values]. / Elévation des lactates-déshydrogénases sériques. Valeur diagnostique, pronostique et évolutive.

OBJECTIVE: Determination of serum lactate dehydrogenase (LDH) levels is an usual practice. However, its place in the diagnosis process is not clear. We have collected serum LDH levels superior to 2-fold the normal rate and we tried to determine their diagnostics interest and, predictive and progressive values. METHODS: Retrospective study during 3 months in hospitalized adults. LDH levels were measured by spectrophotometry (Normal rate: 313-618 UI/L). RESULTS: 196 cases with LDH level elevations higher than 1236 UI/L were analyzed. The etiology of LDH level elevations were was benign in 60% of cases, malignant in 36% and, undetermined in 5%. There was no difference in between average values of LDH level average values of benign and malignant etiology (2708 vs 2842 UI/l). LDH rates and high LDH level elevations were not helpful for in the diagnosis process (a variety of 43 etiology was able to elevate increased LDH rates). In 45% cases, LDH level was 2 to 3-fold the normal rate; in 47.5% cases, 3 to 10-fold normal rate, and in 7.5% cases, superior to 10-fold normal rate. LDH elevations superior to 10-fold the normal rate were caused by benign etiology in 11 cases and malignant disease in 4 cases. A level superior to 10-fold the normal rate was not helpful in determining to determine the benign or malignant characteristics of the initial disease. However, LDH rate superior to 10 normal rate was a pejorative predictive criteria (hospitalization in intensive careunity in 73% of cases and mortality rate of 53%). During follow up of a neoplasia or malignant hemopathy follow up, several LDH measurements LDH level determinations were determined in for a small number of patients. LDH level normalizations is are attributable to efficientan effective treatment; LDH level elevations are associated with a therapeutic failure echappment. Evolution in LDH levels evolution was influenced by progression in neoplasia and malignant hemopathy evolution and also by various several treatments such as like blood transfusions, growth factors, radiotherapy and chemotherapy. CONCLUSION: LDH level elevation, however whatever its rate, don't seem to do not help in differentiating have interest to differentiate benign from malignant diseases. However, an elevation LDH elevation higher than 10-fold the normal rate is a pejorative predictive criteria, since because the mortality rate is superior toupper than 50%. During follow-up of in the neoplasia and malignant hemopathy follow up, so long as they are measured at distance from treatment, variations in LDH levels are a good marker of evolution, rate variations represent an evolutive marker conditionally the level determination would be realize remote several treatment.

Production and certification of secondary enzyme reference materials (ERMs). Part 1: Preparation of the sera and some of their properties.

We produced three batches of a human-serum-based enzyme reference material (ERM) enriched with human aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), creatine kinase (EC 2.7.3.2), and lactate dehydrogenase (EC 1.1.1.27). The added enzymes were not exhaustively purified; thus the final ERMs contained some enzymes as contaminants, of which only glutamate dehydrogenase activity might interfere. The stability during storage and after reconstitution was good. The commutability of the four enzymes in the three ERM batches was also good, except when German or Scandinavian methods for aminotransferases were involved. The temperature-conversion factors for the ERMs were equivalent to those for patients' sera. Reactivation after reconstitution was complete within 5 min and was independent of the temperature of the reconstitution fluid. We believe that these secondary ERMs will aid in the transfer of accuracy between well-defined reference methods and daily working methods so that clinical enzymology results will become more comparable from laboratory to laboratory.

Production and certification of secondary enzyme reference materials (ERMs). Part 2: The need for meaningful protocols that assure photometric accuracy and a well-defined process for value assignment.

A collaborative study to assign values to two enzyme reference materials (ERMs) was performed by 18 laboratories whose spectrophotometers were checked by us, just before the study. We measured the wavelength accuracy and repeatability, the accuracy and linearity of the absorbance curves, the cuvette pathlength, equilibration time, equilibrium temperature, and a few other variables. Five spectrophotometers exhibited a marked wavelength-dependent nonlinearity. Most instruments were rather slow in bringing the sample to the correct temperature and the final temperature was often too high. In the collaborative study, each participant performed the same manual, well-described methods on four occasions in triplicate, using reagents prepared locally. The relation between the photometric checks and the analytical results is discussed, as well as the treatment of outliers and the effects on the variances. Suggestions are made about various facets of collaborative studies. The values assigned to the two ERMs carry a 95% uncertainty interval of +/- 1-4% of the mean.

Purification of lactate dehydrogenase isoenzymes one, two, and three from human erythrocytes.

Lactate dehydrogenase (LD) isoenzymes 1, 2, and 3 were prepared from human erythrocytes by sequential ion-exchange chromatography followed by general-ligand (AMP analog) affinity chromatography. Respective yields, purification factors, and specific activities (kU per gram of protein) were 25%, 4394-fold, and 209.7; 40% 4385-fold, and 199.1; and 18%, 7565-fold, and 192.9. The respective preparations contained less than 0.5% of contaminating LD isoenzyme activity as judged from electrophoresis on thin-layer agarose, were homogeneous as judged by electrophoresis on polyacrylamide gel (both in the presence and absence of sodium lauryl sulfate), and showed minor contamination by other LD isoenzymes as judged by analytical isoelectric focusing. We think that these preparations would be useful as human-based calibrating or reference materials. Their purity is such that these preparations could also be used as antigens for the development of suitable antisera.