Imprecision of cardiac marker analyses among laboratories on the basis of external quality assurance results: Finnish experience.

The purpose of this report was to evaluate the reproducibility and harmonization of cardiac marker tests and to describe the current situation concerning quality of assays for cardiac markers on the basis of the results of the external quality control schemes (EQAS) of Labquality Ltd., Helsinki, Finland in the period 2002 to 2005. Finnish EQAS surveys obtained for proficiency samples at low marker concentration indicated that the overall coefficient of variation (CV) between laboratories for CK-MBmass and troponin I exceeded 10 %, while for cardiac troponin T the CV was 8.6 %. Intra-laboratory reproducibility was investigated in a single laboratory using concomitant testing in the same EDTA plasma samples to establish cut-off limits for one CK-MBmass and three troponin assays. The 10 % imprecision limit obtained from the concomitant testing in the same samples for CK-MBmass was (by Elecsys) 8.5 microg/L, for cardiac troponin T (by Elecsys) 0.023 microg/L and for cardiac troponin I (by AxSYM) and by Immulite 2000) 0.85 microg/L and 0.63 microg/L. At present, it is recommended that laboratories determine the concentration at which the 10 % imprecision for a specific cardiac marker assay is reached, because the assays generally do not reach that imprecision at the level of the 99th percentile value, usually taken as decisional level. However, common efforts of scientific societies and professional diagnostic industry associations internationally are needed if consensus is to be reached on standardization of immunoassays for cardiac markers and uniform results obtained among laboratories.

Best use of the recommended IFCC reference method, material and values in HbA1C analyses.

The results of Finnish HbA(1C) surveys (Labquality Ltd.) during the past 10 years have undergone continuous improvement with smaller overall coefficients of variation for the HbA(1C) mean values of all methods (from 7.5 to 5.4% for normal and from 8.9 to 4.7% for diabetic samples). Most of the HbA(1C) methods are certified for traceability to the Diabetes Control and Complication Trial (DCCT) designated comparison method, which originally was a high-performance liquid chromatography (HPLC) method (Bio-Rex 70, Bio-Rad) but is no longer in routine use. It was therefore important that the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) had prepared both reference preparations and method for the determination of HbA(1C). However, the very demanding reference method is not realistic for use in clinical laboratories. According to the present study, the mean HbA(1C) values of the Labquality Ltd. showed significant correlations to the HbA(1C) values of The European Reference Laboratory for Glycohemoglobin (r = 0.999) and to the values using the IFCC method (r = 0.999). The reference values of the IFCC method (mainly those of the manufacturer) range from 2.85 to 3.81%, being significantly lower than the present DCCT values (4.0-6.1%). Since it may take some time before consumers are ready to accept the new IFCC reference values for general use, we propose that the IFCC reference materials and method should be used for calibration of the present methods to the well-known DCCT levels.

The association of bone metabolism with bone mineral density, serum sex hormone concentrations, and regular exercise in middle-aged men.

Physical activity is an important factor in attaining bone mass. Our aim was to investigate if low to moderate intensity exercise affects bone resorption [serum tartrate-resistant acid phosphatase (TRAP) 5b activity] and formation (serum osteocalcin concentration) in a randomized controlled exercise intervention trial in Finnish middle-aged men. In addition, the relations of these bone turnover markers with bone mineral density (BMD) and serum sex hormone concentrations [circulating testosterone (T), estradiol (E2), and sex hormone-binding globulin (SHBG) concentrations] were evaluated. Serum TRAP 5b activity and osteocalcin concentration were measured at randomization and after 1 and 4 years of the exercise intervention. BMDs of the lumbar spine (L2-L4), femoral neck, and total proximal femur were measured with a dual-energy X-ray absorptiometry (DXA). At randomization, TRAP 5b activity was strongly correlated with the osteocalcin concentration (Spearman r = 0.541, P < 0.0001). In addition, TRAP 5b activity was significantly correlated with proximal femur BMD values (r = -0.201, P = 0.018) and osteocalcin concentration with femoral neck and proximal femur BMD values (r = -0.187, P = 0.028; r = -0.240, P = 0.005, respectively). Serum E2, free E2, and free T concentrations were inversely correlated with both bone turnover markers. After 1 year of exercise intervention, TRAP 5b activity was significantly lower in the exercise than reference group (P = 0.006). However, after 4 years of exercise intervention, the difference was no longer statistically significant. There were no differences in the osteocalcin concentrations between the study groups during the intervention. Our results show a connection between serum TRAP 5b activity and osteocalcin concentration. Furthermore, our results suggest that low to moderate exercise intervention and serum sex hormone concentrations may induce changes in bone metabolism in middle-aged men. However, exercise-induced effects on bone metabolism should be confirmed in other randomized controlled exercise trials taking into account exercise intensity and dose-response issues.

Aerobic exercise and bone mineral density in middle-aged finnish men: a controlled randomized trial with reference to androgen receptor, aromatase, and estrogen receptor alpha gene polymorphisms.

Our aim was to investigate associations of the polymorphic loci of androgen receptor (AR), aromatase CYP19, and estrogen receptor alpha (ERalpha) genes with bone mineral density (BMD) in a four-year controlled randomized exercise intervention trial in Finnish middle-aged men. Additionally, we studied whether the gene polymorphisms affect circulating testosterone (T), estradiol (E(2)), and sex hormone-binding globulin concentrations. The polymorphic CAG repeat of the AR gene, the TTTA repeat of the human aromatase gene, and the PvuII site of the ERalpha gene were analyzed. BMDs of the lumbar spine (L2-L4), femoral neck, and total proximal femur were measured with a dual-energy X-ray absorptiometry (DXA). In the exercise group, the subjects with the ERalpha gene PP or Pp genotypes showed an increase (+6.5 and +5.1%, respectively) in lumbar spine BMDs (P = 0.007; repeated measures ANOVA) during intervention, while there was no change in the subjects with the pp genotype. The long TTTA repeat (TTTA(9-12)) in aromatase gene was associated with greater height (P = 0.026) and lower BMI (P = 0.029) values than the short TTTA repeat (TTTA(6-8)). With regard to the AR gene, no statistically significant differences in bone properties were found between the genotypes. There were no significant associations of any analyzed polymorphic sites with the serum sex steroid hormone concentrations in the exercise or reference group. In conclusion, the Finnish middle-aged men with ERalpha PP or Pp genotypes appear to have increased BMD values in the lumbar spine. This increase may reflect a predisposition to age-related degenerative changes in the spine. In addition, the AR CAG repeat and aromatase TTTA repeat do not modify the effect of regular aerobic exercise on BMD.

Stromelysin-1 and interleukin-6 gene promoter polymorphisms are determinants of asymptomatic carotid artery atherosclerosis.

The functional 5A/6A polymorphism of the stromelysin-1 promoter has been implicated as a potential genetic marker for the progression of angiographically determined atherosclerosis in patients with coronary artery disease. Recently, a novel interleukin-6 (IL-6) gene functional G/C polymorphism at -174 in the promoter has also been reported. In this study, we analyzed the relation of these two polymorphisms with carotid artery atherosclerosis in 109 randomly selected, middle-aged men without exercise-induced ischemia. Atherosclerosis was quantified as intima-media thickness (IMT) by high-resolution ultrasonography. Univariately, stromelysin genotype was significantly (P:=0.015) associated with IMT, and this relation remained (P:=0.033) after adjustments for age, cardiorespiratory fitness, body mass index, smoking, LDL cholesterol, and systolic blood pressure and for sonographers. The 5A/6A polymorphism independently explained 7% of the variance in carotid bifurcation IMT. The IL-6 polymorphism was also significantly associated (P:=0. 036) with increased IMT, with men homozygous for the G allele having IMT that was 11% greater than men homozygous for the C allele. Men who were homozygous for both the 6A and G alleles had an covariate adjusted IMT that was 36% greater than men who were homozygous for neither allele (P:<0.003). These data suggest that genetic factors that predispose to reduced matrix remodeling (stromelysin 6A allele) and to increased inflammation (IL-6 G allele) combine to increase susceptibility for intima-media thickening in the carotid bifurcation, a predilection site for atherosclerosis.

The nutritional status of Finnish home-living elderly people and the relationship between energy intake and chronic diseases.

The nutritional status and the impact of non-progressive chronic diseases on energy intake were determined in 90 home-living people aged from 73 to 94 years. The nutritional status was assessed by dietary, anthropometric, biochemical and haematological methods. Energy intake (6.0, SD 1.7 MJ) in women was low compared with the Nordic Nutrient Recommendation but in men it (8.0, SD 2.1 MJ) was in keeping with this recommendation. Despite the low energy intake the mean BMI value of women was moderately high (27, SD 5.3 kg/m2). In men the mean was 26, SD 4.0 kg/m2. The intakes of vitamins and minerals met the recommendation, except for those of folic acid and zinc. The blood levels of both these two nutrients were within reference limits. Men suffering from chronic diseases received less (p < 0.015) energy (7.5, SD 1.76 MJ) than other men (8.9, SD 2.0 MJ). This relationship was not found in women. In conclusion, the nutritional status of people aged over 70 years old living at home was good. The presence of chronic diseases affected the energy intake in men but not in women.

Inverse relation of physical activity and apolipoprotein AI to blood pressure in elderly women.

To study the relation of habitual physical activity, diet, and serum lipoproteins to blood pressure, a cross-sectional study was carried out in a cohort of 202 women, age 60-69 yr. Sitting, supine, and standing blood pressure was measured with a standard sphygmomanometer. Physical activity was assessed by questionnaire, diet by food records, serum lipoprotein cholesterol enzymatically, and apolipoprotein AI turbidimetrically. Among the women not taking antihypertensive medication (N = 127), the physically most active (physical activity 5 times per week or more) had sitting diastolic blood pressure of 86 mm Hg (adjusted for high-density-lipoprotein cholesterol, body mass index, and cardiovascular health status), which was 8 mm Hg lower (P = 0.007) than in the least active (physical activity twice per week or less) women. Subjects in the highest tertile of apolipoprotein AI (> 1.46 g.l-1) had a mean sitting systolic blood pressure of 147 mm Hg (adjusted for age, body mass index, and cardiovascular health status), which was 16 and 13 mm Hg lower (P = 0.001) than in women in the middle and lowest tertiles (< 1.32 g.l-1), respectively. The present data suggest that, in elderly women, regular physical activity is associated with a clinically significant lowering of diastolic blood pressure. Moreover, a higher level of serum apolipoprotein AI, the major protein component of high-density lipoprotein particles, is inversely associated with systolic blood pressure.

Flow cytometric method to measure urea-resistant alkaline phosphatase from blood neutrophils for use in the prenatal screening of Down's syndrome.

The histochemical measurement of urea-resistant alkaline phosphatase from maternal blood neutrophils is known to have a high detection rate for the prenatal detection of Down's syndrome pregnancies. However, because the histochemical method is laborious and subjective to use, it has not gained widespread acceptance in prenatal screening programmes. We present a simple and objective method for the measurement of urea-resistant alkaline phosphatase by flow cytometry. The method should allow the design of larger studies aimed at evaluating the role of neutrophil urea-resistant alkaline phosphatase in the prenatal screening for Down's syndrome.

Relation of habitual diet and cardiorespiratory fitness to blood coagulation and fibrinolytic factors.

The relation of habitual diet and cardiorespiratory fitness to plasma fibrinogen concentration, Factor VII activity (F VIIc), Factor X activity (F X), tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1) concentrations, anti-thrombin III (AT III) and apolipoprotein(a) (Apo[a]) was analyzed in 111 normolipidemic men aged 51-53 years. Diet was evaluated by seven day food records. Maximal oxygen consumption and aerobic threshold were determined in maximal bicycle ergospirometry test based on breath-by-breath analysis of expired respiratory gas. Plasma fibrinogen was measured by thrombin method, F VIIc by one-stage coagulation method, AT III and F X colorimetrically, t-PA and PAI-1 antigens by ELISA and Apo(a) concentration radioimmunologically. Carbohydrate intake was negatively (r = -0.31, p < 0.001; r = -0.24, p < 0.01; r = -0.36, p < 0.001) and fat intake positively (r = 0.24, p < 0.01; r = 0.29, p < 0.001; r = 0.32, p < 0.001) related to F X, PAI-1, and t-PA, respectively. Aerobic threshold correlated negatively with fibrinogen (r = -0.33, p < 0.001) and F X (r = -0.30, p < 0.001). Fasting insulin was the strongest determinant for PAI-1 (r = 0.55, p < 0.001) and a significant positive correlate to F VIIc (r = 0.30, p < 0.001), F X (r = 0.28, p < 0.01) and t-PA (r = 0.47, p < 0.001). These data emphasize the importance that carbohydrate rich diet and cardiorespiratory fitness may have against thrombogenesis.

Quality specifications for glycated hemoglobin A1c.

Berne has suggested that a change in the measured value (in per cent) of GHbA1c that is smaller than 1.0% does not lead to any therapeutic action from the clinician. Since the meeting of the "Friibergh Herrgård Seminar", April 23-24, 1990 on the "Medical Need for Quality Specifications in Laboratory Medicine" we have discussed this with our specialists at the Kuopio University Hospital and they share Berne's opinion. Keeping this in mind we have standardized our method so that the total analytical variation of the GHbA1c is small as compared to the measured biological variation of GHbA1c in nondiabetic and diabetic subjects. In this report we describe how we have succeeded in keeping the coefficient of variation of our routine method for GHbA1c analyses small enough for clinical purposes.

Quality specifications for S-AFP and S-HCG-beta determinations for screening of fetal abnormalities.

High levels of maternal serum alpha-1-fetoprotein (S-AFP) are associated with fetal structural abnormalities like neutral tube defects and congenital nephrosis. It has a very high detection rate when used for screening of these diseases. On the other hand, the low levels of maternal S-AFP act as a marker of many fetal chromosomal aberrations. The detection rate of S-AFP alone for the screening of Down's syndrome is low. Wald et al. have shown that the addition of the determination of maternal serum chorionic gonadotrophin (S-HCG-beta) to S-AFP analysis, and using a computer program for risk estimation increases the detection rate for Down's syndrome to about 57% with a false positive rate of 5%. In this study we present the results of our screening program for structural fetal defects and chromosomal aberrations in Eastern Finland, and describe the standardization and quality control of maternal serum AFP and HCG-beta analysis.

Low serum vitamin D metabolites in women with rheumatoid arthritis.

The etiology of osteoporosis associated with rheumatoid arthritis (RA) is unknown. We studied the calcium and vitamin D metabolism in 143 women with RA (mean age 50.7 years). Albumin corrected serum calcium was normal. Serum alkaline phosphatase was increased in 29 percent of cases. Serum vitamin D levels were frequently very low. In 16 percent of the RA patients serum 25(OH)D concentration was below 12.5 nmol/L, which is arbitrarily considered as the limit of vitamin D deficiency osteomalacia. In the winter season 73 percent of the patients had serum 1,25(OH)2D levels below the seasonally adjusted normal range. The lowest values were found in patients with high disease activity. We suggest that there is a disturbance in vitamin D metabolism in RA. This might play a role in osteoporosis associated with RA.

Comparison of poly- and monoclonal antibodies for determination of B-cell clonal excess in an routine clinical laboratory.

Flow cytometry (FCM) has gained wide use in the determination of clonality in B-cell lymphoproliferative diseases and many methodological variations exist. We have compared the suitability of a) dual fluorochrome (FITC/PE)-labelled monoclonal antibodies, b) single fluorochrome (FITC)-labelled monoclonal antibodies and c) F(ab')2 fragments of FITC-labelled polyclonal antibodies for flow cytometric determination of clonality using commercially available software and a short sample preparation protocol. The FCM method was validated by analysis of immunoglobulin heavy chain and light chain gene rearrangements. We recommend the use of FITC-labelled monoclonals to obtain three parameters, the kappa/lambda ratio, D and D/S(n) values (Kolmogorov-Smirnov statistics) instead of the commonly used kappa/lambda ratio and D values only. This allows the use of a rapid sample preparation protocol to blood and bone marrow aspirates without sacrificing sensitivity or specificity obtained by the usual FCM method.

Detection and quantitation of sperm-bound antibodies by flow cytometry of human semen.

Detection of sperm surface antibodies is important for the diagnosis and treatment of infertility. The authors have investigated the methodologic aspects of flow cytometry (FCM) to detect sperm-bound antibodies and to quantitate the sperm antibody load (antibody molecules/spermatozoa). To obtain reliable results, dead spermatozoa must be excluded from analysis because they can bind antibody nonspecifically, and comprise 10% to 58% (n = 28) of the ejaculate in subfertile men. Flow cytometry estimation of dead cells correlates (r = 0.83) significantly with the manual Eosin Y method. After staining washed sperm samples (n = 26) with fluorescein-isothiocyanate-conjugated F(ab')2 fragments of anti-IgG and IgA antibodies, the sperm load was 11,500 +/- 8,600 IgG molecules and 13,200 +/- 9,500 IgA molecules per spermatozoa. The sperm antibody load measured on different occasions could be compared between patients or in the same patient by the use of calibration standards. Since the inter- and intra-assay variation of the FCM assays was less than 10%, FCM has the potential reliably and objectively to monitor the sperm antibody load during corticosteroid treatment.

The European Stroke Prevention Study: results according to sex.

The European Stroke Prevention Study was a multicenter trial comparing the effect of a combination of 75 mg dipyridamole and 330 mg acetylsalicylic acid tid with placebo in the prevention of stroke or death after one or more attacks of recent transient ischemic attacks or stroke of atherothrombotic origin. From the 2,500 patients in the intention-to-treat analysis, the proportion of women was 42%, and from the 1,861 patients in the explanatory analysis it was 44%. The endpoint incidence was significantly higher in men than in women. The endpoint reduction was statistically significant only in the intention-to-treat analysis with total endpoints. However, there was a marked percentage reduction of endpoints in both men and women in explanatory analysis. The risk reduction of strokes was 49% for men and 41% for women, and the reduction of total endpoints was 39% in men and 30% in women. Thus, antiplatelet therapy is effective in the prevention of stroke or death in both sexes.

Use of quality control standards in clinical flow cytometry.

Flow cytometry is important in the clinical laboratory, especially in hematology and cancer diagnosis. With newer applications being continuously developed clinicians who will use the results and laboratory staff who produce them should be aware of the instrumental quality control procedures required. This will allow evaluation of the reliability as well as the comparison of results between different laboratories. While information about sample preparation as well as staining procedures is easily available, information about instrumental quality control procedures is less so. Various standards and controls are available commercially and their correct choice and usage requires an understanding of the methodology of instrumental quality control. Methods to evaluate the precision, sensitivity and accuracy of measurements are discussed. Most laboratories report immunophenotyping data as the percentage of antibody positive cells and make no attempt to quantitate the amount of antibody binding per cell. Methods for quantitating the number of bound antibody molecules per cell along with a simple technique to compare results between laboratories are discussed. These techniques should encourage laboratory staff and clinicians to standardise methodology and to exchange patient data between laboratories.

Dose-response relationships in blood lipids during moderate freshwater fish diet.

Fish and fish oils are known to counteract coronary heart disease risk factors. We have previously showed that eating moderate amounts of freshwater fish modifies lipid and prostanoid metabolism in healthy students. In this study, the dose response relationship was clarified. One hundred male students took part and were randomly divided into control and four fish diet groups with different fish diets. Hematological, serum lipid and vitamin E and A analyses were performed. Already 1.5 fish containing meals per week increased n-3 to n-6 ratio of fatty acids in erythrocyte ghost phosphatidylethanolamine. The serum triglyceride and apolipoprotein B concentration fell significantly in the group eating 3.8 fish containing meals a week for 12 weeks. At two lower doses (1.5 and 2.3 meals/week) the tendency to lower values was already seen. No significant changes were observed in the serum cholesterol, apolipoprotein A1 and vitamin E and A concentrations. The hematological variables also remained unchanged. The results show that moderate amounts of fish as a constituent of normal diet have beneficial effects on lipid metabolism.

Effect of drugs on histamine radio-enzyme assay.

The effect of over 200 drugs and other compounds on histamine radio-enzyme assay was studied. Some muscle relaxants (e.g. alcuronium), some sympathomimetics (e.g. dopamine, isoxsuprine, tyramine and possibly phenylethylamine), antimalarial drugs, procaine, procainamide, Berenil and serotonin were potent compounds to interfere with this assay. In some special cases still potentially inhibitory drugs seemed to be some muscle relaxants (e.g. vecuronium, pancuronium and tubocurarine), antidepressants, antihistamines (e.g. cimetidine, ranitidine and diphenhydramine), chinidin, disopyramide, tolazoline and salazosulfapyridine.