Volume 38, Issue 3

The enzyme activities of creatine kinase (CK), its isoenzyme MB (CK-MB) and of lactate dehydrogenase isoenzyme 1 (LD-1) have been used for years in diagnosing patients with chest pain in order to differentiate patients with acute myocardial infarction (AMI) from non-AMI patients. These methods are easy to perform as automated analyses, but they are not specific for cardiac muscle damage. During the early 90's the situation changed. First creatine kinase MB mass (CK-MB mass) replaced the measurement of CK-MB activity. Subsequently cardiac-specific proteins troponin T (cTnT) and troponin I (cTnI) appeared on the scene, displacing LD-1 analysis. However, troponin concentrations in blood increase only from four to six hours after onset of chest pain. Therefore a rapid marker such as myoglobin, fatty acid binding protein or glycogen phosphorylase BB could be used in early diagnosis of AMI. On the other hand, CK-MB isoforms alone may also be useful in rapid diagnosis of cardiac muscle damage. Myoglobin, CK-MB mass, cTnT and cTnI are nowadays widely used in diagnosing patients with acute chest pain. Myoglobin is not cardiac-specific and therefore requires supplementation with some other analyses such as troponins to support the myoglobin value. Troponins are very highly cardiac-specific. Only the sera of some patients with severe renal failure, which requires hemodialysis, have elevated cTnT and /or cTnI without there being any evidence of cardiac damage. On the other hand, the latest studies have shown that elevated troponin levels in sera of hemodialysis patients point to an increased risk of future cardiac events in a similar manner to the elevated troponin values in sera of patients with unstable angina pectoris. In addition, the bedside tests for cTnT and cTnI alone or together with myoglobin and CK-MB mass can be used instead of quantitative analyses in the diagnosis of patients with chest pain. These rapid tests are easy to perform and they do not require expensive instrumentation. For routine clinical laboratory practice we suggest that in diagnosis of patients with chest pain, myoglobin and CK-MB mass measurements should be performed whenever they are requested (24 h/day) and cTnT or cTnI on admission to the hospital and then 4–6 and 12 hours later.

Total homocysteine is the sum of all homocysteine species present in plasma. This parameter is valuable for the diagnosis and follow up of cobalamine or folate deficiencies, and it has been accepted as an independent risk factor for premature cardiovascular disease. These reasons justify the introduction of total homocysteine assays in the routine clinical laboratory. In the present review, we discuss the procedures for sample collection, processing and storage, and review the methodologies for measuring total homocysteine. We compare their feasibility as routine methods in the clinical laboratory.

The serum protein designated 90K/Mac-2BP has been found at elevated concentrations in the sera of patients with various types of cancer and viral infections. The importance of the 90K/Mac-2BP serum concentrations in predicting the response towards interferon-α treatment for hepatitis C virus (HCV) infection prompted us to utilize a new ELISA for soluble human 90K/Mac-2BP to monitor the serum concentrations of this protein in our HCV-positive patients. Seventy HCV-PCR and anti-HCV antibody positive patients were analyzed for their serum levels of aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase, cholinesterase, HCV-viral load, viral subtypes, and 90K/Mac-2BP. On correlation of age and 90K/Mac-2BP levels, we found an apparent correlation that was proved rather to be a strong dependence of 90K/Mac-2BP concentrations on disease severity/duration, which increases with age. Multiple correlation analysis demonstrated the independent nature of 90K/Mac-2BP concentrations, underscoring the potential high utility of this new marker. Our data corroborate the potential of the scavenger receptor family protein 90K/Mac-2BP as an independent predictor of disease severity during HCV infection.

To assess whether interferon β1a (IFNβ1a) therapy affects plasma lipoprotein metabolism, twelve patients with relapsing-remitting multiple sclerosis (MS) were studied during a two-year follow-up period. High density lipoprotein (Hdl 2 ) cholesterol and the Hdl 2 /Hdl 3 ratio were increased at year 2 and lipoprotein (a) was transitorily increased at year 1, in comparison to baseline levels. Apolipoprotein A-I was lower and apolipoprotein E higher at year 1, only in a subgroup of patients who experienced relapses and/or progressed during therapy. These findings suggest that IFNβ1a treatment is associated with changes in the lipoprotein metabolism. Alterations in this metabolism could be related to the immunomodulatory actions of the drug and the disease activity in multiple sclerosis patients.

An experimental model was developed to study the kinetics of electrolytes under different physiological and/or pathological conditions. The model was applied to investigate in vivo the effect of a pharmacological dose of melatonin on the concentrations of Ca 2+ , K + , Na + , and pH in the anticoagulated blood of anaesthetized male Wistar rats (250–350 g). After the application of 0.25 mg melatonin/kg body weight, injected intraperitoneally into each of 8 rats, the electrolytes were measured by a flow-through system with highly sensitive ion-selective electrodes. The results were compared to a control group (n=8) which was treated with diluent (saline). The electrolytes were monitored continuously via an extracorporeal circulation, on-going for at least 60 min. Melatonin induced a significant increase of blood Ca 2+ (p<0.02) by an average of 9.9% after 60 min. However, total calcium concentration did not increase significantly. The extracorporeal circulation provoked an elevation of K + by hemolysis. This K + increase was slightly diminished by melatonin (p<0.06). No melatonin effects were seen on Na + , pH and magnesium in blood and plasma, respectively. Also, the urine concentrations of the electrolytes were not altered by melatonin. The mechanism by which melatonin influences the blood concentrations of ionized calcium and potassium is not yet understood.

The aim of this study was to investigate the biochemical and hematological changes in patients on routine hemodialysis treatment when they were accidentally exposed to moderately high serum aluminum concentrations during a period of time of less than four months. We studied the changes in biochemical and hematological measurements in 33 patients on dialysis in our hospital before and during the exposure to about 0.85 μmol/l of aluminum in dialysis water due to a malfunction of the reverse osmosis system of water purification. Patients showed a decrease in the hemoglobin concentration from 115 ± 12.4 g/l to 108 ± 12.2 g/l (p=0.026) and in the mean corpuscular hemoglobin concentration from 5.15 ± 0.22 to 5.02 ± 0.30 mmol/l (p=0.014). Ferritin was decreased from 243 ± 192 μg/l to 196 ± 163 μg/l (p= 0.047) and transferrin saturation from 0.20 ± 0.06 to 0.15 ± 0.07 (p= 0.004). Biochemical measurements related to calcium-phosphate metabolism did not change. Otherwise, all patients showed an increase in serum aluminum from 0.56 ± 0.44 to 1.63 ± 0.52 μmol/l (p<0.001). No differences were detected in serum aluminum between patients receiving and not receiving oral aluminum salts. Even moderately high aluminum concentrations maintained during a short period of time could produce significant hematological alterations and a depletion of body iron stores before clinical manifestations were evident.

We studied the accuracy and precision of a HPLC method for determination of porphyrins in urine. A commercial standard solution appeared to contain less porphyrins than indicated by the manufacturer, since calibration resulted in lower concentrations of uroporphyrin and coproporphyrin: 16% and 8%, respectively. Coefficients of variation for the measurement of uro-, hepta-, copro I and copro III porphyrins in samples of patients with and without porphyria were often much less than 15%. Comparison of measurements with and without calibrated standards revealed differences for uroporphyrin and coproporphyrin of 27% and 5%, respectively. Recovery of added uroporphyrin and coproporphyrin was 99%. The main cause of the variability in test results was apparently the improperly calibrated standard solutions. The precision of porphyrin measurements was not influenced by the type of porphyria.

Heart-type fatty acid-binding protein (H-FABP) is a low molecular weight cytoplasmic protein and present abundantly in the myocardium. When the myocardium is injured, as in the case of myocardial infarction, low molecular weight cytoplasmic proteins including H-FABP are released into the circulation and H-FABP is detectable in a blood sample. We have already developed a direct sandwich-ELISA for quantification of human H-FABP using two distinct types of monoclonal antibodies specific for human H-FABP. In this study we investigated the clinical validity of H-FABP as a biochemical diagnostic marker in the early phase of acute myocardial infarction (AMI). To evaluate the diagnostic usefulness of H-FABP in the early phase of AMI, blood samples were obtained from the following patients within 12 hours after the appearance of symptoms, and serum levels of H-FABP were compared with those of conventional diagnostic markers, such as myoglobin and creatine kinase isoenzyme MB (CK-MB). Blood samples were collected from patients with confirmed AMI (n=140), patients with chest pain who were afterwards not classified as AMI by normal CK-MB levels (non-AMI) (n=49) and normal healthy volunteers (n=75). The serum concentration of H-FABP was quantified with our direct sandwich-ELISA. The concentration of myoglobin mass was measured with a commercial RIA kit. The serum CK-MB activity was determined with an immuno-inhibition assay kit. The overall sensitivity of H-FA B P, within 12 hours after the appearance of symptoms, was 92.9%, while it was 88.6% with myoglobin and 18.6% with CK-MB. The overall specificity of H-FABP was 67.3%, while it was 57.1% with myoglobin and 98.0% with CK-MB. The diagnostic efficacy rates with these markers were 86.2% (H-FABP), 80.4% (myoglobin) and 39.2% (CK-MB), respectively. The diagnostic validity of H-FABP was further assessed by receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) of H-FABP was 0.921, which was significantly greater than with myoglobin (AUC: 0.843) and CK-MB (AUC: 0.654). These parameters, such as sensitivity, specificity, diagnostic efficacy and diagnostic accuracy, obtained for patients with chest pain within 3 hours and/or 6 hours after the onset of symptoms were almost the same as those for patients within 12 hours after symptoms. H-FABP is more sensitive than both myoglobin and CK-MB, more specific than myoglobin for detecting AMI within 12 hours after the onset of symptoms, and shows the highest values for both diagnostic efficacy and ROC curve analysis. Thus, H-FABP has great potential as an excellent biochemical cardiac marker for the diagnosis of AMI in the early phase.

A procedure is described for the determination of the affinity constant between a fluid-phase biotinylated antigen and a solid-phase monoclonal antibody. This procedure allows evaluation of the efficiency of an antibody as a coated tool for an immunoassay. For this purpose, the biotinylation of the antigen and its further quantitative measurement by streptavidin-peroxidase led to a single reversible interaction, the binding affinity of which greatly determines the quality of the assay. The free and bound fractions of the biotinylated antigen were obtained in wells coated with a low level of immobilized antibodies. At the equilibrium state, the free antigen present in the supernatant of these wells was further transferred to high level antibody coated wells which captured all the free antigen molecules. These molecules were quantified using a standard curve established with known concentrations of biotinylated antigen, also incubated in wells coated with the high level of antibody.

Determination of reticulocytes in peripheral blood is a valuable tool for getting information about erythropoiesis of an individual. For many years, reticulocyte numbers were quantified manually by means of a microscope after staining with supravital dyes. However, this method is tedious and shows low reproducibility. Therefore, several methods for the automated determination of reticulocytes have been established in laboratory routine within the last years. The aim of this study was to compare three of these automated methods for reticulocyte analysis. Reticulocytes were determined in 130 subsequent routine samples by means of an ABX Pentra 120 Retic haematological analyser, a Coulter EPICS XL MCL flow cytometer and a Coulter STKS haematology system, using the fluorescent dye thiazole orange or the supravital dye new methylene blue for reticulocyte staining, respectively. The reticulocyte concentrations were slightly lower for the Coulter STKS haematology system (mean ± SD 1.89 ± 1.32%) when compared with the Coulter EPICS XL MCL flow cytometer or the ABX Pentra 120 Retic haematological analyser (2.11 ± 1.25% and 2.12 ± 1.15%, respectively). The correlations between all methods were significant (r s ≥ 0.843, p < 0.001). Small intercepts were, however, observed in the correlation plots between the values obtained by means of the Coulter STKS haematology system and those obtained by the other two methods. Within-batch coefficients of variation were 6.0%, 6.9% and 7.8% for the ABX Pentra 120 Retic haematological analyser, the Coulter STKS haematology system and the Coulter EPICS XL MCL flow cytometer, respectively. The corresponding between-batch coefficient of variation values were 6.8%, 4.9% and 5.3% as well as 14.1%, 7.6% and 6.1% for the low, medium and high control levels determined by means of the ABX Pentra 120 Retic haematological analyser and the Coulter STKS haematology system, respectively. These data suggest that all three methods allow the efficient and reliable determination of reticulocyte counts under clinical routine conditions. However, although the obtained data are very similar, differences exist which should be taken into account for the normal values of the different methods.

Thyrotropin (TSH), free thyroxine (fT4) and testosterone assays have been used as a probe to evaluate the performances of a new modular chemiluminescence (CL) immunoassay analyser, the Abbott Architect™ i 2000. The evaluation was run in parallel on other systems that use CL as the detection reaction: DPC Immulite ® , Chiron Diagnostics ACS-180 and ACS Centaur (TSH functional sensitivity only). TSH functional sensitivity was 0.0012, 0.009, 0.033 and 0.039 mU/l for the Architect, Immulite, ACS Centaur and ACS-180, respectively. Testosterone functional sensitivity was 0.38, 3.7 and 2.0 nmol/l for Architect, Immulite and ACS-180, respectively. Good correlation was obtained between the ACS-180 and Architect for all assays. The Immulite correlation did not agree well with the Architect or ACS-180 for fT4 and testosterone but was in good agreement for TSH. Regarding fT4 and testosterone, equilibrium dialysis and isotopic dilution gas-chromatography mass-spectrometry (GC-MS) respectively were used as reference methods. For both within- and between-run precision, the Architect showed the best reproducibility for all three analytes (CV < 6%).

In the recent issue of CCLM a paper has been published on the view of the German Working Group on Point-of-Care Testing (POCT) (1). The issue of POCT is extremely important and rightly the authors stress the responsibility of the head of the laboratory. However, they omit to refer to two important documents relating to POCT from the European Communities Confederation of Clinical Chemistry (EC4): one on the essential criteria for quality systems of medical laboratories (2) and another on the additional essential criteria for quality systems of medical laboratories (3), the latter addressing in particular POCT. Both of these documents strongly influence new ISO documents on quality management in medical laboratories. The section of the German Working Group's paper on organizational prerequisites for the establishment of POCT in a hospital could particularly benefit from these references.