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Tritiated thymidine labelling in vitro of human cancer of the breast: counting error and sampling error.

Tritiated thymidine labelling indices (TLIs) were determined on a number of primary cancers of the human breast. Twenty-two slides were chosen which demonstrated a wide range of TLIs and each was counted twice to assess 'counting error'. TLIs derived from successive counts of the same slide showed a coefficient of variance greater than 25% in half of the slides, but a significant difference between the two (P less than 0.05) in only 2 of 22 pairs. When TLIs derived from paired specimens taken from different sites in each of 22 tumours were compared, there was found to be a significant difference (P less than 0.05) in 17 of 22 pairs. This is 'sampling error' and is clearly a major source of inaccuracy when TLIs are derived from single small samples of heterogeneous tumours such as cancers of the human breast.

Breast Neoplasms

Sampling errors in pH and blood gas analysis--an evaluation of three new arterial blood samplers.

We have tested the accuracy, acceptability and general performance of three recently-marketed samplers for arterial blood gas measurement (the Corning Arterial Blood Sampler, the Concord 'Pulsator' and the Sarstedt 'Monovette'). All three greatly reduce or eliminate the error of venous sampling, and the Corning and Sarstedt samplers eliminate the risk of dilution of the sample by excess heparin solution. A positive bias in pO2 measurement, more marked at higher levels, was demonstrated with the Concord and Sarstedt samplers, and the latter carry a slightly increased risk of cross-infection. None of the samplers completely overcame potential sampling errors.

Arteries

A microcomputer program for evaluating sampling error: an application to stereological methods for electron microscopy.

In situations where there is a need to minimize sampling error or sample size, the coefficient of variation (CV) may be used to evaluate sampling error as a function of the number of observations or subjects in a sample. For example, CV is useful for estimating the minimum number of electron micrographs (Nmin) required to obtain a representative field sample for stereological analysis. To facilitate the determination of Nmin, we have written a program (COEFficient) for DOS microcomputers which calculates CVs. COEF assists the user in reducing error to that which solely reflects biological variability, thereby minimizing the time and cost of subsequent analyses.

Microcomputers

Variability in muscle fibre areas in whole human quadriceps muscle: how to reduce sampling errors in biopsy techniques.

A single biopsy is a poor estimator of the muscle fibre cross-sectional area (CSA) for a whole human muscle because of the large variability in the fibre area within a muscle. To determine how the sampling errors in biopsy techniques can be reduced, data on the CSA of type 1 and type 2 fibres obtained from cross-sections of whole vastus lateralis muscle of young men, have been analysed statistically. To obtain a good estimate of the mean fibre CSA in a biopsy, measuring all fibres in that biopsy gives the best result. To obtain a good estimate of the mean fibre CSA for a whole muscle, the number of biopsies has a much greater influence on the sampling error than the number of fibres measured in each biopsy, but the number of biopsies needed to obtain a given sampling error can vary by a factor of two. If the fibre CSA in three or more biopsies is measured, it is sufficient to measure only 25 fibres in each biopsy. If less than three biopsies are taken, there is no worthwhile reduction in sampling error when more than 100 fibres are measured. To determine the mean fibre CSA for a whole group of individuals, our preference is to maximize the number of individuals, and only take single biopsies. In conclusion, to determine the mean fibre CSA for this particular muscle with a certain precision, we suggest analysis of three biopsies, taken from different depths of the muscle, and measurement of 25 fibres in each biopsy.

Adolescent

Subhourly variability of circulating third trimester maternal steroid concentrations as a source of sampling error.

Seven plasma samples from five normal third trimester pregnant women, drawn every 5 and 15 min at similar times on 2 days, 2 days apart, were measured in quadruplicate for estradiol (E2), estriol, progesterone, 16 alpha-hydroxyprogesterone, and 17 alpha-hydroxyprogesterone (17P). The mean of the 22 samples obtained from each subject for each steroid was used as a reference mean. Individual determinations were converted to percentages of the reference mean which was normalized to 0%. All pregnancies were uncomplicated, and all mean values were within the normal range for gestational age. Variability about the reference mean for single samples, however, range from a low of -80% to a high of 150%. The single sample, 95% confidence intervals for individual steroids pooled from the five subjects (110 determinations) range from +/- 36% (E2) to +/- 60% (17P). Mean percentage coefficients of variation between 5-min and 15-min sampling sequences were compared by analysis of variance. There is no significant difference between the mean percentage coefficients of variation of a 5-min as opposed to a 15-min sampling sequence for any of the hormones measured. The 95% confidence interval width around the reference mean is a function of the number of samples obtained. Because the 95% confidence interval width from 110 measurements decreases approximately as 1/ square root n with increasing sampling size, the decrement progressively diminishes. For E2, the least variable steroid, a one-sample 95% confidence interval width of +/- 36% decreases to approximately +/- 18% with four samples or approximately +/- 12% with nine samples. For 17P, the most variable steroid, a one-sample 95% confidence interval width of +/- 60% decreases to approximately +/- 30% with four samples or approximately +/- 20% with nine samples. Multiple sampling with plasma pooling is required for the accurate study of steroid concentrations in individual subjects in late pregnancy.

17-alpha-Hydroxyprogesterone

Analysis of sampling errors in biopsy techniques using data from whole muscle cross sections.

Because of the large variability in the proportion of fiber types within a whole muscle, a single biopsy is a poor estimator of the fiber type proportion for a whole muscle. Data on the proportions of type I and II fibers, obtained from cross sections of whole human muscles (vastus lateralis) from young male individuals, have therefore been analyzed statistically in order to determine the sampling errors involved in muscle biopsy techniques. For the purpose of obtaining a good estimate of the fiber type proportion in a whole biopsy, counting all fibers is of great benefit compared with counting only half of the fiber number. The required number of biopsies to obtain a given sampling error of the mean proportion of fiber types in the whole muscle can vary by a factor of six. If less than three biopsies are taken from a muscle, there is a substantial reduction in sampling error taking biopsies with at least 600 fibers. For more than three biopsies there is a small gain in sampling greater than 150 fibers. The precision of the estimate of the mean proportion of fiber types for a group is increased with the number of biopsies per individual and number of individuals. In conclusion, for the muscle in this study, complete counting of three biopsies, each greater than 150 fibers, sampled from different depths of the muscle is recommended.

Adolescent

A morphometrical comparison of right and left whole human vastus lateralis muscle: how to reduce sampling errors in biopsy techniques.

In studies of the effects of different training programmes, one muscle--most commonly the vastus lateralis--is used for the experiment while the contralateral muscle serves as a control, at the same time as muscle biopsies are taken from both sides. In order to increase the reliability of such studies, the sources and the magnitude of the sampling errors in the biopsy techniques need to be assessed in detail. In this study, cross-sections of whole right and left vastus lateralis muscle from six young sedentary right-handed men were prepared, and the total number and size of fibres and the proportion of the different fibre types were calculated. A significant difference (P less than 0.05-P less than 0.001) between the right and the left muscle was found for at least one of the three variables in each of the six men, but there was no systematic difference and, therefore, no significant right-left difference for the whole group. The maximum difference between the right and the left side for the mean fibre size was 25% and for the fibre type proportion 5%; these differences are much smaller than the known variation within individual muscles. In conclusion, any study involving biopsies from both the right and the left vastus lateralis may use either muscle for the experiment while the contralateral muscle serves as a control without leading to systematic sampling error, whereas the errors involved in taking small samples from each muscle are much more important to control and to reduce.

Adult

Sampling error in diagnosing hyperparathyroid changes in bone in small needle biopsies.

Sampling error in diagnosing the effects of hyperparathyroidism on bone was studied retrospectively by subdividing large-core bone biopsy samples and evaluating them histologically. We used intratrabecular osteoclastic tunneling resorption as the diagnostic feature of secondary hyperparathyroidism in 10 hemodialysis patients. All biopsies were taken with a large-core bone biopsy instrument, resulting in a mean biopsy width of 5 mm. The processed tissue slides were divided into halves (mean width 2.5 mm) and subsequently into thirds (mean width 1.6 mm), mimicking smaller bone biopsy instruments such as the Jamshidi needle. In 30% of the cases the diagnosis of secondary hyperparathyroidism would have been missed if taken by a Jamshidi-type needle, and 60% if taken by a needle with a core diameter of 1.6 mm. Our findings emphasize the necessity of utilizing large-core bone biopsies in diagnosing certain metabolic bone diseases.

Biopsy, Needle

Evaluation of postmortem endomyocardial biopsy specimens from 38 patients with lymphocytic myocarditis: implications for role of sampling error.

Among 38 hearts from autopsies in which lymphocytic myocarditis contributed to death, 10 endomyocardial specimens from the apical septal aspect of each ventricle (760 specimens) and 6 slices of ventricular myocardium (228 slices) were evaluated for myocarditis by the Dallas criteria. For each case, the number of positive biopsy samples correlated well with the mean lymphocyte counts in biopsy tissues (P less than 0.0001) and the mean number of inflammatory foci per square centimeter in myocardial slices (P less than 0.001). Right ventricular biopsy specimens, however, were positive in only 63% of the 38 cases and 17% of the 380 specimens. Similarly, left ventricular biopsy tissues were positive in only 55% of the cases and 20% of the specimens. Sampling error was somewhat more prevalent among the 11 cases with isolated myocarditis than in the 27 with myocarditis and other illnesses. Even when 10 biopsy specimens per ventricle were evaluated, the frequency of false-negative results was 45% for the left and 37% for the right ventricle. Although myocarditis was noted in 68% of the 38 septal slices, it involved the subendocardium of the right ventricle (from which biopsy specimens are usually obtained) in only 24%. Because of the mild and focal nature of the inflammatory infiltrates and involvement of regions inaccessible to the bioptome, sampling error contributes appreciably to false-negative results in endomyocardial biopsy tissue from patients with myocarditis. Thus, when myocarditis is evaluated by biopsy alone, only positive findings are considered diagnostic.

Adolescent

Sampling error and observer variation in the interpretation of esophageal biopsies.

Esophageal autopsy specimens from 11 subjects were used to determine observer variation and sampling error in assessing alterations attributed to gastroesophageal reflux. Inter- and intraobserver variation exceeded 20% even when the diagnosis was limited to a normal and abnormal reading. Marked differences in basal cell thickness but not in papillary height occurred when specimens were obtained from different levels of the lower esophagus. The differences were less marked in specimens obtained from the same level. Based on these data the reliability of basal cell thickness and papillary height as an index of gastroesophageal reflux appears limited.

Autopsy

Sampling errors and the precision associated with counting very low numbers of white cells in blood components.

Attention to the accurate and precise measurement of the white cell (WBC) content of transfused products has risen in response to awareness of the potential benefits of WBC-depleted components and the development of technical capabilities to produce these components. The techniques thus far reported have focused on the reliability of detecting a WBC, provided it is present in the test system. The likelihood of selecting a WBC from the product of interest for counting in the analytical system--that is, the sampling error--must also be considered. The occurrence of a WBC in a WBC-depleted component is a rare event and may be modeled with the binomial or the Poisson distribution. Several assay techniques were analyzed by using these distribution models to determine the confidence intervals of the WBC content. The 95-percent confidence intervals spanned more than 2 logs10 for some methods at 3 x 10(5) WBCs per product. It is concluded that the reporting of WBC content for research provides not only the estimate of the mean but also a confidence interval for this estimate. Quality control procedures should be designed to verify that the WBC content is less than the targeted amount and should provide an associated statement of confidence.

Blood Platelets

Variations in the apparent nutrient content of foods: a study of sampling error.

1. Successive portions of boiled and mashed potatoes, roast pork, cooked, freeze-dried peas and ice-cream were taken in order to determine the total (sampling and experimental) error involved in the 'duplicate analysis' method of dietary survey. 2. These samples were analysed for water, nitrogen, fat, iron and energy. 3. The experimental error of the methods was also studied separately. 4. The sampling and experimental errors ranged from 0-3% for water in potatoes, to 23% for Fe in ice-cream. Sampling error was significantly greater than experimental error for all nutrients studied. 5. It is concluded that in reporting the results from dietary surveys, it is necessary to take account of the erros inherent even in the most precise methods.

Analysis of Variance

Heterogeneity of lung cancer: the problem of sample error in diagnostic electron microscopy.

We studied the ultrastructure of superficial and deep samples of 40 resected primary lung carcinomas. Tumour cell differentiation was semiquantitatively assessed and differences between samples of a same tumour were evaluated. In two instances were major differences in ultrastructural diagnosis found between the samples of the same tumour. A further 9 cases showed one predominant differentiation in one sample, but two equally predominant differentiations in the second sample. The other 29 tumours did show occasional minor differences between the samples, but these differences did not result in differences in ultrastructural diagnosis.

Carcinoma, Small Cell

Computer-assisted human semen analysis. Sampling errors and reproducibility.

Videomicrographic computer-automated semen analysis systems allow quantitative description of sperm motility, velocity, progression, and head movement amplitude and frequency with unprecedented ease. The minimum number of spermatozoa needed for stable results, the variability of measurements and optimum methods of sampling the ejaculate were determined for one such system (Cell-Soft, CRYO Resources, New York, NY). Sampling a minimum of 225 spermatozoa yields stable measurements, and analyzing four microscope fields in triplicate provides data with the lowest coefficient of variation. The variability attributable to the instrument itself was acceptable for all measurements (6.2% to 15.4%) except mean amplitude of lateral head displacement. Limitations of these results and the potential utility of videomicrographic sperm movement analysis are discussed.

Electronic Data Processing

Discrepancies in ploidy determination due to specimen sampling errors.

Two techniques are described to enhance the detection of low frequency aneuploid cells in automated cell analysis. One method concerns a cell preparation technique; the other is focused on specific cell selection at the measurement level. The cell preparation method has been designed to select and process the tumour areas in paraffin blocks and can be used for image as well as for flow cytometry. The technique uses incident fluorescence microscopy for visual inspection of the surface of the fluorescently stained tissue block to select the specific tumour parts. Using image cytometry, it is shown that in tissue sections with very small tumour foci and many normal cells, aneuploidy could only be detected after enrichment of the cell sample with the specifically selected areas. The cell selection at the measurement level is directed towards detection of low frequency aneuploid cells on microscope slides using the specific capacities of LEYTAS (Leyden Television Analysis System). With this system, cells of interest can be selected by means of minimum size and intensity thresholds. In addition to measurement of the total cell population, all cells above a minimum DNA value can thus be specifically selected and measured. The advantage of both enrichment techniques is the possibility to detect and measure aneuploid cell lines in cases where normal, diploid cells dominate the paraffin tissue.

Aneuploidy

Campylobacter pylori infection in biopsy specimens of gastric antrum: laboratory diagnosis and estimation of sampling error.

Campylobacter pylori infection was sought in 382 consecutive patients referred for upper gastrointestinal endoscopy. Five antral biopsy specimens were taken from each patient: one was inserted into a CLO-test to detect the urease activity of C pylori, two were sent for histological analysis where multiple sections were stained by the Warthin-Starry silver method, and two were sent for microbiological evaluation by Gram stain and culture. A patient was deemed to be infected when C pylori was cultured or seen in either the histological sections or the Gram stain of the biopsy smear. One hundred and seventy four (46%) patients were infected. Culture, Gram stain, histological examination and the CLO-test showed sensitivities of 92%, 87%, 93% and 90%, respectively. In 27 (15%) infected patients an uneven distribution of C pylori was seen between samples in the biopsy pair sent for histology. Examination of multiple sections stained with Warthin-Starry silver was more sensitive at detecting infection (93%) than examination of multiple sections from only one biopsy specimen (84%). Fifty seven of 80 patients, biopsied a median seven days (range 5 to 55) after completing colloidal bismuth subcitrate treatment, were still infected with C pylori. There was no decrease in the sensitivities of the above tests to detect infection after treatment. It is concluded that at least two antral biopsy specimens should be examined when attempting to diagnose C pylori infection by histological methods.

Biopsy