Ole Blaabjerg

Analytical goals for the acceptance of common reference intervals for laboratories throughout a geographical area

Analytical goals required for the successful transfer of reference intervals between laboratories within a specified limited geographical area, with a population homogeneous for the quantities, are presented. Diagrams are shown which allow the investigation of the influence of analytical imprecision and bias, both separately and in combination, on the percentage of the population outside each reference limit. Figures to evaluate the effect of population sample size on the size of confidence interval around each reference limit are combined with the diagrams for analytical imprecision and bias. The maximum acceptable percentage of the population outside the limit for the 0.90 confidence interval of each of the means +/- 1.96 s reference limits is 4.6% for a population sample size of 120. Based on this, the maximum acceptable imprecision, with no bias, is 0.6 of the total biological standard deviation (sB) and the maximum acceptable bias, with no imprecision, is 0.25 sB.

Establishment of a serum thyroid stimulating hormone (TSH) reference interval in healthy adults. The importance of environmental factors, including thyroid antibodies

Abstract It has previously been shown that thyroid antibodies affect thyroid stimulating hormone (TSH) concentrations in men and women and that TSH levels are predictive of future thyroid disease. We investigated the validity of the National Academy of Clinical Biochemistry (NACB) guidelines regarding the TSH reference interval by studying 1512 individuals. Two hundred and fifty had at least one thyroid antibody, 121 were taking medications other than estrogens and occasional analgesics, and 105 reported a family history of thyroid disease. Serum TSH, thyroid peroxidase antibodies (TPOab) and thyroglobulin antibodies (Tgab) were determined on AutoDELFIA and TSHRab by a radioreceptor assay (RRA) from Brahms Diagnostica. For individuals without thyroid antibodies and other risk factors, no effect of age and gender was seen for serum TSH. Neither medication nor the presence of Tgab alone had any influence on serum TSH. TPOab alone or in combination with Tgab were associated with an increased serum TSH level. The ‘cumulative percentage distributions’ of subgroups, as well as the combined population, was ln-Gaussian distributed. The central 95% of the population was within the 95% CI in rankit-plots. Consequently, a common reference interval for serum TSH of 0.58–4.07 mIU/l for all adults between 17 and 66 years of age was established. This reference interval is much higher than expected from the NACB-guidelines.

Screening for haemochromatosis: prevalence among Danish blood donors

Abstract. Hereditary haemochromatosis is an autosomal recessive disease that is genetically expressed by excessive accumulation of iron in the tissues, resulting in cirrhosis, diabetes mellitus, cardiomyopathy and hypogonadism. As the disease may be diagnosed before the appearance of symptoms, and prevented by repeated phlebotomies, there are strong implications for adoption of a screening procedure. Determinations of transferrin saturation (TS) and serum ferritin concentration (SF) were used to screen 4302 blood donors, who were selected for follow‐up studies if they fulfilled any of the following three criteria: (i) TS ≥ 0.7; (ii) TS ≥ 0.5 together with SF ≥ 150 μg l−1; (iii) SF ≥ 300 μg l−1. A total of 58 subjects who fulfilled at least one of these criteria were reinvestigated, after which 18 individuals still fulfilled at least one criterion. Fifteen subjects having SF ≥ 300 μg l−1 were offered liver biopsy and thirteen of these accepted. In one individual, no stainable iron was detected, and two subjects did not fulfil the previously established diagnostic criteria for the diagnosis of hereditary haemochromatosis. Ten subjects who had a high TS and liver iron grade 2–4 according to Bassett were classified accordingly as homozygotes. On the basis of these results, the prevalence of haemochromatosis in Denmark was estimated to be 0.0037–0.0046.

Establishment of reference distributions and decision values for thyroid antibodies against thyroid peroxidase (TPOAb), thyroglobulin (TgAb) and the thyrotropin receptor (TRAb).

Abstract Background: The National Academy of Clinical Biochemistry (NACB) stresses that the reference intervals for thyroid peroxidase antibodies (TPOAb), thyroglobulin antibodies (TgAb) and thyroid stimulating hormone (TSH)-receptor antibodies (TRAb) should be based on young men who lack certain risk factors and have serum TSH between 0.5 and 2.0mIU/L. However, some young men without any of the risk factors have autoantibodies, and cannot be identified by the present tools. A model for reference intervals and cut-off values should not be influenced by the prevalence of risk factors. Methods: We developed a model of “composite logarithmic Gaussian distributions” and tested it in 1441 well-characterised subjects without clinically overt thyroid disease. Results: TPOAb and TgAb could be measured in all individuals. The 97.5% upper limits 1) on a traditional non-parametric scale, 2) according to the NACB criteria, and 3) for our model were 284, 24 and 9.8kIU/L for TPOAb, and 84, 22 and 19kIU/L for TgAb, respectively. The decision value (defined as the concentration corresponding to 0.1% false positives) was 15kIU/L for TPOAb and 31kIU/L for TgAb. Concentrations above our reference intervals affected the corresponding distribution of TSH values. For TRAb the upper reference limits were 1) 0.75 and 2) 0.75IU/L, while our model was not applicable to TRAb because only 2–3% of the results were above the functional assay sensitivity. Conclusions: In contrast to the NACB guidelines, our model for TPOAb and TgAb is more robust, as it is independent of the characteristics of the reference population. Clin Chem Lab Med 2006;44:991–8.

Analytical Goals for the Estimation of Non-Gaussian Reference Intervals

Goals for analytical quality are postulated for the situation in which a number of laboratories will share common reference intervals for some quantities for which the population is homogeneous. These goals are evaluated on the assumption that the acceptable combined analytical bias and imprecision should not allow the percentage of the population outside either upper or lower reference limits to exceed the interval 1.3 to 4.4%. The goals are evaluated for log-Gaussian biological distributions, and for distributions which can be transformed into log-Gaussian by addition of a constant value to all elements. Further analytical goals for non-parametric distributions are discussed. For log-Gaussian distributions the maximum allowable bias and imprecision are dependent on the ratio of the upper and lower reference limits. When this ratio is c. 1.25, the bias alone must be less than 1.5% or the coefficient of variation alone less than 3.5%. For a ratio of 10, these values may be as high as 15% and 34%, respectively.

An enzyme-linked immunosorbent assay for plasma-lactoferrin. Concentrations in 362 healthy, adult blood donors

Very different concentrations of plasma-lactoferrin in healthy adults have been reported in the literature. We compared three commercially available lactoferrins and lactoferrin purified in our laboratory as calibrators in an ELISA. No statistical differences among these preparations of lactoferrin were detected. The concentration of purified lactoferrin was measured by dry weight, and efforts were made in order to minimize loss of purified lactoferrin by adhesion to tubes etc. and thus, secure accuracy of the method. Dilutions were made in PBS 0.01 mol l-1 with NaCl 0.436 mol l-1, (NH4)2SO4 0.5 mol l-1, BSA 5 gl-1 and normal rabbit IgG 10 mg l-1, which was shown to give parallel dilution curves of primary calibrator, secondary calibrator and plasma samples. No significant difference in the content of lactoferrin in neutrophils (median; range) among men (1.78; 0.83-4.48 micrograms 10(-6) neutrophils; n = 20) and women (2.12; 1.16-9.30 micrograms 10(-6) neutrophils; n = 14) was found. Lactoferrin was analysed in EDTA-plasma obtained from 135 female and 227 male blood donors. Median concentrations were 84.7 and 97.8 micrograms l-1 respectively, while 2.5% and 97.5% reference limits (with 90% confidence intervals) were estimated to 42.9 (38.7-47.4) micrograms l-1 and 166.9 (151.0-186.3) micrograms l-1 for women and 52.3 (49.1-55.6) micrograms l-1 and 189.9 (175.9-206.4) micrograms l-1 for men, respectively.

Graphical interpretation of confidence curves in rankit plots

Abstract A well-known transformation from the bell-shaped Gaussian (normal) curve to a straight line in the rankit plot is investigated, and a tool for evaluation of the distribution of reference groups is presented. It is based on the confidence intervals for percentiles of the calculated Gaussian distribution and the percentage of cumulative points exceeding these limits. The process is to rank the reference values and plot the cumulative frequency points in a rankit plot with a logarithmic (ln=loge) transformed abscissa. If the distribution is close to ln-Gaussian the cumulative frequency points will fit to the straight line describing the calculated ln-Gaussian distribution. The quality of the fit is evaluated by adding confidence intervals (CI) to each point on the line and calculating the percentage of points outside the hyperbola-like CI-curves. The assumption was that the 95% confidence curves for percentiles would show 5% of points outside these limits. However, computer simulations disclosed that approximate 10% of the series would have 5% or more points outside the limits. This is a conservative validation, which is more demanding than the Kolmogorov-Smirnov test. The graphical presentation, however, makes it easy to disclose deviations from ln-Gaussianity, and to make other interpretations of the distributions, e.g., comparison to non-Gaussian distributions in the same plot, where the cumulative frequency percentage can be read from the ordinate. A long list of examples of ln-Gaussian distributions of subgroups of reference values from healthy individuals is presented. In addition, distributions of values from well-defined diseased individuals may showup as ln-Gaussian. It is evident from the examples that the rankit transformation and simple graphical evaluation for non-Gaussianity is a useful tool for the description of sub-groups.

Serum lactate dehydrogenase isoenzyme 1 and tumour volume are indicators of response to treatment and predictors of prognosis in metastatic testicular germ cell tumours

44 patients with metastatic testicular germ cell tumours treated with cisplatin-based chemotherapy were evaluated for prognostic implications of clinical characteristics. 22 obtained complete remission by the initial chemotherapy, and 30 are disease-free. S-LDH-1 had an overall predictive value regarding the response of 80%, S-LDH of 64%, S-AFP of 62%, and S-hCG of 62%. In multivariate analysis regarding response, only tumour volume classified according to the Royal Marsden system (P = 0.0036) and S-LDH-1 (P = 0.0069) yielded information. Regarding survival, S-LDH-1 (P = 0.0141) and an estimate of total tumour mass (P = 0.0171) had most impact with additional information from S-hCG only (P = 0.0536). We conclude that S-LDH-1 may be used as a tumour marker in addition to S-hCG and S-AFP in patients with metastatic testicular germ cell tumour.

Serum lactate dehydrogenase isoenzyme 1 as a marker of testicular germ cell tumor

Serum lactate dehydrogenase isoenzyme 1 activity was determined repeatedly in 21 men with testicular germ cell tumors in connection with orchiectomy and in 25 without neoplasia who underwent exploration of the testis. The highest level in the men without malignancy was 109 units per 1. and a higher pre-orchiectomy level was found in 15 of the tumor patients: 7 of 11 with seminoma and 8 of 10 with nonseminomatous tumors. In the patients with stage 3 disease serum lactate dehydrogenase isoenzyme 1 was increased more often and the activity was higher than in the stage 1 and 2 cancer patients. Within 1 month after orchiectomy the initially increased level decreased to less than 109 units per 1. in 8 of 10 patients with a stage 1 tumor and it remained higher in 5 with stage 2 or 3 disease. Serum lactate dehydrogenase isoenzyme 1 seems to be a useful marker of testicular germ cell tumor.

Analytical quality specifications for serum lactate dehydrogenase isoenzyme 1 based on clinical goals

Abstract The aim of the study was to deduce analytical quality specifications for the determination of catalytic concentration of serum lactate dehydrogenase isoenzyme 1 (S-LD-1) according to clinical goals (the clinical utility model). We defined clinical goals for false positive and false negative S-LD-1 measurements in the monitoring of patients with testicular germ cell tumors (TGCT), clinical stage I, on a surveillance only program. The absolute S-LD-1 catalytic concentrations were routinely corrected for contamination from preanalytical hemolysis. A reference group of 37 men had a near ln-Gaussian distribution for the absolute S-LD-1 catalytic concentration. The geometric mean was 76 U/l and an S-LD-1 > 128 U/l (99.72 percentile, the decision limit) indicated a high risk of a relapse of TGCT. We have previously shown that an S-LD-1 > 160 U/l (treatment limit) was associated with a suboptimal outcome from the treatment of metastatic TGCT. The maximum allowable analytical positive bias was 5 U/l, and the maximum allowable analytical negative bias was −32 U/l. The maximum allowable analytical coefficient of variation, CVA, was 11 % (≈14 U/l) at a bias = −5 U/l. For S-LD-1 measurements not corrected for hemolysis, the decision limit was 145 U/l, the maximum allowable negative bias −19 U/l, and CVA 8 % (≈12 U/l). A routine correction for hemolysis had a large impact on the analytical quality specifications.