A review of amyloid staining: methods and artifacts

Abstract

Amyloid detection is very precise at this time, because several methods are available to the pathologist. Awareness of potential nonspecific staining, possible pitfalls and methods for improving the detection process are basic to enhancing the staining of amyloid and interpreting this staining. The role of the pathologist has progressed through history from the basic detection of amyloid as a substance to immunophenotypical classification of the particular amyloid present.     

Statistical methods for the practical clinical application of morphometric measurements

Four statistical methods are presented to determine the practical clinical value of measurements made from malignant tumors and to translate these measurements into a prediction of survival for each patient: the Cox statistical model, which must be derived from a data base of cases with known outcome; the null-rank test, a modified rank-sum test that provides an overall measure of the effectiveness of the Cox model; the predicted survival curve, an estimate of survival derived for each new patient from measurements of the primary tumor; and the standard error of measurement, an empirical method for estimating the variability introduced into predicted survival by errors in measurement. The value of these statistical methods was demonstrated by application to 200 cases of human intraocular melanoma, with the two predictive morphometric measurements used being the standard deviation of nucleolar area (SDNA) and the largest tumor dimension (LTD) derived from a single histologic slide of each tumor. Sufficient references and mathematical details are provided to allow anyone with moderate skills as a computer programmer to construct or obtain all of the relevant algorithms.     

Reproducibility and variation of morphometric analysis of carcinoembryonic antigen staining in histopathology. The influence of standardized microscopic fields

Using morphometric methods, five pathologists analyzed the positive staining for carcinoembryonic antigen (CEA) in sections from 17 ovarian tumors, with the intraclass correlation coefficient (ICC) and the mean values of the coefficients of variation (CV) used to assess reproducibility and variation. First, field and point scores for epithelium and mucin were estimated using 25 randomly selected square fields in sections from each of the tumors. The ICC range in the whole sample field was 0.53 to 0.81 (slight to substantial reproducibility) while the mean values of CV were 0.50 to 0.75. Second, the results of using random and standardized individual fields for the measurements were studied in three tumors. In random fields, the ICC was 0.57 to 0.71 (slight to moderate reproducibility) and the mean values of CV were 0.53 to 0.65. The corresponding values in standardized fields were 0.71 to 0.73 (moderate reproducibility) and 0.41 to 0.57, respectively. The results show that the variation is smaller and the degree of reproducibility higher in standardized fields. Considerable variation remains, however, revealing human factors as an important source of variation in practical morphometry.     

Reproducibility of nuclear morphometric measurements in benign human epithelial cells. Simulation of variations in routine tissue processing.

The value of nuclear morphometric measurements in diagnostic pathology is determined largely by the reproducibility of the measurements. Although a variety of factors have been shown to affect tissues during processing, the regulation of fixative type and the avoidance of air drying in particular have been shown to avoid significant variations in nuclear measurements. The current study simulated routine tissue processing in order to identify any factors that may introduce variability of nuclear morphometric values in day-to-day processing if air drying is avoided and fixative type and pH are regulated. Samples of benign endometrium were collected from three uteri, fixed in phosphate-buffered formalin (PBF) from 2 hours to 15 days and dehydrated in an automated tissue processor on four occasions. In addition, tissue from one case was cut at 4, 6 and 8 microns, simulating the potential variations in section thickness that may occur during routine processing. Mean nuclear areas and shape factors of epithelial cells were then determined using computed planimetry. By analysis of variance, no significant differences were found in nuclear morphometric values in relation to time of fixation, dehydration runs or tissue section thickness; coefficients of variation for all variables were less than 7%. This study suggested that routinely processed tissues are adequate for morphometric analysis, including retrospective analysis, provided that tissues are fixed in a pH-regulated fixative such as PBF and air drying is avoided.     

Reproducibility of electronic tooth colour measurements

Clinical methods of investigation, such as tooth colour determination, should be simple, quick and reproducible. The determination of tooth colours usually relies upon manual comparison of a patient's tooth colour with a colour ring. After some days, however, measurement results frequently lack unequivocal reproducibility. This study aimed to examine an electronic method for reliable colour measurement. The colours of the teeth 14 to 24 were determined by three different examiners in 10 subjects using the colour measuring device Shade Inspector. In total, 12 measurements per tooth were taken. Two measurement time points were scheduled to be taken, namely at study onset (T(1)) and after 6 months (T(2)). At either time point, two measurement series per subject were taken by the different examiners at 2-week intervals. The inter-examiner and intra-examiner agreement of the measurement results was assessed. The concordance for lightness and colour intensity (saturation) was represented by the intra-class correlation coefficient. The categorical variable colour shade (hue) was assessed using the kappa statistic. The study results show that tooth colour can be measured independently of the examiner. Good agreement was found between the examiners.     

Reproducibility of non-fasting plasma metabolomics measurements across processing delays

Introduction

Processing delays after blood collection is a common pre-analytical condition in large epidemiologic studies. It is critical to evaluate the suitability of blood samples with processing delays for metabolomics analysis as it is a potential source of variation that could attenuate associations between metabolites and disease outcomes.

Objectives

We aimed to evaluate the reproducibility of metabolites over extended processing delays up to 48 h. We also aimed to test the reproducibility of the metabolomics platform.

Methods

Blood samples were collected from 18 healthy volunteers. Blood was stored in the refrigerator and processed for plasma at 0, 15, 30, and 48 h after collection. Plasma samples were metabolically profiled using an untargeted, ultrahigh performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) platform. Reproducibility of 1012 metabolites over processing delays and reproducibility of the platform were determined by intraclass correlation coefficients (ICCs) with variance components estimated from mixed-effects models.

Results

The majority of metabolites (approximately 70% of 1012) were highly reproducible (ICCs?≥?0.75) over 15-, 30- or 48-h processing delays. Nucleotides, energy-related metabolites, peptides, and carbohydrates were most affected by processing delays. The platform was highly reproducible with a median technical ICC of 0.84 (interquartile range 0.68–0.93).

Conclusion

Most metabolites measured by the UPLC–MS/MS platform show acceptable reproducibility up to 48-h processing delays. Metabolites of certain pathways need to be interpreted cautiously in relation to outcomes in epidemiologic studies with prolonged processing delays.

     

Methods for staining amyloid in tissues: a review

The traditional way of identifying amyloid in tissue sections has been staining with Congo red and demonstration of green birefringence under crossed polarizers. The original method of Congo red staining, described by Bennhold in 1922, has undergone several modifications to improve its sensitivity, specificity, and reliability. The most common modification is the alkaline Congo red method described by Puchtler and co-workers in 1962. Specificity is improved by using freshly prepared stain and a staining solution fully saturated with sodium chloride. Amyloid proteins can be further distinguished by autoclaving or by treating the tissue with potassium permanganate or alkaline guanidine. Autoclaving the tissues at 120 C for 30 min causes protein AA to lose its affinity for Congo red. Prolongation of autoclaving to 120 min abolishes the Congophilia of protein AL, but prealbumin-related amyloid shows little or no change. Treatment of the tissue with potassium permanganate causes protein AA and B2-microglobulin amyloid to lose their affinity to Congo red. Protein AA fails to stain with Congo red after treatment with alkaline guanidine for 1 min and protein AL and systemic senile amyloid protein (SSA) after 2 hr. Familial amyloid protein (FAP), prealbumin type, can stand 2 hr of alkaline guanidine treatment without losing its ability to stain with Congo red. Other methods of detection of amyloid include fluorescent stains, e.g., thioflavin T or S, and metachromatic stains such as crystal violet. Immunofluorescence and immunoperoxidase methods are used to identify and classify amyloid proteins in tissues. Antibodies against the P component, proteins AA and AL and FAP have been used with great precision. Due to cross-reactivity, these methods do not differentiate between some types of familial and senile systemic amyloidosis.     

Reproducibility of the biosynthetic parameters in various fermentation equipment

The final parameters of tetracycline biosynthesis in different fermentation apparatus were analysed comparatively with an account of possible changing of the fermentation broth volume against the initial one due to water evaporation. It was shown that for comparison of the biosynthesis parameters in different fermentation apparatus it is necessary to reestimate the activity values or the antibiotic concentration in a unit of the medium volume with respect to the initial volume of the fermentation medium charged into the apparatus.     

Demonstrating morphometric protocols using polystome marginal hooklet measurements

The flexible body structure of polystomes (Monogenea: Polystomatidae) encumbers taxonomic classification and species identification. Large intraspecific and limited interspecific variation in the morphology of polystomes further complicates the identification of species. Apart from employing the host-specific nature of the polystomes, taxonomic characterisation relies heavily on the sclerotised skeletal structures, such as the hamuli and the marginal hooklets. The currently accepted measurement system for marginal hooklets appears to be non-optimal and an improved protocol is needed. This paper describes in detail how such a protocol is found by evaluating various sets of measurements statistically in order to identify the most informative combination of parameters. Thirteen measurements of marginal hooklets from 11 different Southern African Polystoma species were taken and evaluated. A new protocol for discriminating between species of Polystoma Zeder, 1800, that employs only three measurements, is proposed. The value of the processes to derive morphometric protocols, as described, is that it is not restricted to a specific taxon, but that it can be amended and applied to any taxonomic grouping.     

Predicting biomass of Atlantic salmon from morphometric lateral measurements

Previously biomass predictions have been derived from simple weight—length relationships. This study measured a variety of truss and conventional dimensions covering the lateral body profile of Atlantic salmon Salmo salar and, using regression analysis, developed a series of multifactor weight—lateral dimension relationships. Single-factor regression equations proved inadequate for predicting weight with percentage errors between real and estimated values ranging from −1.2±6.8% to 72.5 ± 225.6%. Fifty-two multifactor regression equations were generated that predicted accurately the weight of individual fish to within ±2% using combinations of conventional and truss measurements. Regression coefficients were found to be significantly different ( P <0.05) between Scottish and Norwegian strains, indicating morphological differences between the genetic groups. Norwegian fish were generally heavier for a given length compared to Scottish strains. This suggests that morphologically different strains of S. salar would require individual weight: lateral dimension relationships to be developed in order to predict biomass accurately to within commercially acceptable levels.     

Regeneration of the rat sciatic nerve after various experimental lesions (morphologic and morphometric analysis)

Dynamics of structural restoration of the peripheral nerve (n. ischiadicus) have been studied in the noninbred rats in 3 series of experiments: after local freezing, pinching and cutting with a subsequent connection of the nerve ends by means of an implanted arterial vessel. As demonstrate the methods of light and electron microscopy, myelinization of the nervous fibers in the distal part of the nerve begins between the 10th-20th days after the effect. Further, amount of the myelinated nerve fibers (NF) significantly increases, they become essentially thicker. However, even in the later time of the observation (9 months) most of NF remain thinner than in the control nerve; this demonstrates that the reparative processes take a longer time than it was supposed before. The comparative analysis makes it possible to recommend the cryogenic lesion of the nerve as the most perspective model to study processes of the reparative histogenesis. Certain positive signs of sutureless connection of the cut nerve by means of the implanted arterial vessel are noted for clinical substitution of vast diastases of the nerve.     

Statistical power comparisons among alternative morphometric methods

This paper compares the statistical power of various tests that have been proposed to test for equality of shape in two populations. Power surfaces are computed with emphasis on the simplest case of three points in the plane (i.e., landmarks at the vertices of a triangle). Goodall's ([1991] J Roy Stat Soc Serb 53:285-339) F-test was found to have the highest power followed by T(2)-tests using Kendall tangent space coordinates. Power for T(2)-tests using Bookstein shape coordinates was good if the baseline was not the shortest side of the triangle. The Rao and Suryawanshi ([1996] Proc Natl Acad Sci 93:12132-12136 and Rao and Suryawanshi [1998] Proc Natl Acad Sci 95:4121-4125) shape variables had much lower power when triangles were not close to being equilateral. Power surfaces for the EDMA-I T statistic revealed very low power for many shape comparisons including those between very different shapes. Power surface for the EDMA-II Z statistic were also complicated and depended strongly on the choice of baseline used for size scaling. The type I error rate was also often not correct for this method. Results for more than three landmarks are also presented. The implications of the results for practical applications of morphometrics are discussed.     

Reproducibility of anthropometric measurements in children: a longitudinal study

The purpose of this study was to establish the reproducibility of a series of anthropometric measures performed twice during one week during a three year period in boys and girls. The subjects of this investigation were 39 children (21 boys and 18 girls), 9-10 year of age at the beginning of the study. Children were measured three times with one year interval. Children were classified by Tanner stage 1-2 during the first measurements, stage 1-3 during the second measurements and stage 1-4 during the third measurements. Body height and weight were measured and BMI calculated. All anthropometric parameters were measured according to the protocol recommended by the International Society for the Advancement of Kinanthropometry (Norton & Olds 1996). Nine skinfolds, 13 girths, eight lengths and eight breadths/lengths were measured. The reproducibility of body height (r = 0.995-0.999), body weight (r = 0.990-0.999) and BMI (r = 0.969-0.999) was very high in boys and girls. The intraclass correlations (ICC), technical errors (TE) and coefficients of variation (CV) were quite different depending on the measurement site of the skinfold thickness. It was surprising that the ICCs were highest and TEs and CVs were lowest during the second year of the measurement. The computed ICC was high, and TE and CV values were quite similar and relatively low in girth, length and breadth/length measurements. It was concluded that the reproducibility of girths, lengths and breadths/lengths in children is very high and the reproducibility of skinfolds is high. Specifically, the reproducibility is very high immediately before puberty in boys and girls.     

Reproducibility and age-related changes of ocular parametric measurements in rabbits

ABSTRACT: BACKGROUND: The rabbit is a common animal model for ophthalmic research, especially corneal research. Ocular structures grow rapidly during the early stages of life. It is unclear when the rabbit cornea becomes mature and stabilized. We investigated the changes of keratometry, refractive state and central corneal thickness (CCT) with age. In addition, we studied the intra- and inter-observer reproducibility of anterior chamber depth (ACD) and anterior chamber width (ACW) measurements in rabbits using anterior segment-optical coherence tomography (AS-OCT). RESULTS: The growth of New Zealand White rabbits (n = 16) were monitored from age 1 to 12 months old. Corneal keratometric and refractive values were obtained using an autorefractor/keratometer, and CCT was measured using an AS-OCT. Keratometry and CCT changed rapidly from 1 to 7 months and appeared to be stabilizing after 8 months. The reduction of corneal curvature was approximately 1.36 diopter (D)/month from age 1 to 7 months, but the change decelerated to 0.30 D/month from age 8 to 12 months. An increase of 10 mum/month in CCT was observed from age 1 to 7 months, but the gain was reduced to less than 1 mum/month from age 8 to 12 months. There was a hyperopic shift over the span of 12 months, albeit the increase in spherical equivalent was slow and gradual. Rabbits of random age were then selected for 2 repeated ACD and ACW measurements by 2 independent and masked observers. Bland-Altman plots revealed a good agreement of ACD and ACW measurements inter- and intra-observer and the ranges of 95% limit of agreement were acceptable from a clinical perspective. CONCLUSIONS: Corneal keratometry, spherical equivalent refraction and CCT changed significantly during the first few months of life of rabbits. Young rabbits have been used in a large number of eye research studies. In certain settings, the ocular parametric changes are an important aspect to note as they may alter the findings made in a rabbit experimental model. In this study, we have also demonstrated for the first time a good between observer reproducibility of measurements of ocular parameters in an animal model by using an AS-OCT.     

Sexing sooty terns on Ascension Island from morphometric measurements

Sooty terns Onychoprion fuscata are one of the most abundant seabirds but breeding populations in many colonies have diminished. Rapid sexing of sooty terns in the field could be crucial in advancing our understanding of their reproductive biology, and in promoting conservation. However, sooty tern males and females are identical in their plumage and, thus, difficult to sex in the field. Morphometric measurements were taken from 63 adult sooty terns breeding on Ascension Island in 2005. A small blood sample was taken from the brachial vein to determine the bird's sex using standard PCR-based molecular techniques. Males were consistently larger in all morphometric measurements than females but considerable overlap between the sexes resulted in no single measurement being a useful discriminator of sex. A principal components analysis on a correlation matrix of seven morphometric measurements indicated that the first principal component (PC1) was a good 'body size' axis explaining 40.5% of the variance in the original matrix. The suite of head measurements all had high character loadings on PC1 and were, therefore, good indicators of the body size of sooty terns. Tarsus length and wing length were less reliable predictors of sex. Discriminant analyses revealed that a disciminant function incorporating head measurements and wing length allowed 77.8% of sooty terns to be sexed correctly based upon morphometric measurements alone. Further morphometric approaches to sexing should be explored with sooty terns captured in subsequent years.     

Sensitivity and nonspeicific staining of various immunoperoxidase techniques

Optimally fixed paraffin enbedded tissue sections and cytocentrifuged cell smears were used to test the sensitivity and nonspecific staining with the enzyme-bridge, PAP, indirect and direct immunoperoxidase methods using human immunoglobulins and lysozyme as antigens. With the enzyme-bridge method positive staining was seen with primary antiserum dilutions up to 1:20,000. The least background staining was observed with this method. The PAP method was equally sensitive, although false-negative results with low primary antiserum dilutions were seen. Some nonspecific background staining always persisted using the PAP method even with high primary antiserum dilutions. The indirect method was not as sensitive as the enzyme-bridge method and some nonspecific staining always persisted. The direct method was too insensitive with paraffin embedded tissue sections.     

Sensitivity and nonspecific staining of various immunoperoxidase techniques

Summary Optimally fixed paraffin embedded tissue sections and cytocentrifuged cell smears were used to test the sensitivity and nonspecific staining with the enzyme-bridge, PAP, indirect and direct immunoperoxidase methods using human immunoglobulins and lysozyme as antigens. With the enzyme-bridge method positive staining was seen with primary antiserum dilutions up to 1:20,000. The least background staining was observed with this method. The PAP method was equally sensitive, although false-negative results with low primary antiserum dilutions were seen. Some nonspecific background staining always persisted using the PAP method even with high primary antiserum dilutions. The indirect method was not as sensitive as the enzyme-bridge method and some nonspecific staining always persisted. The direct method was too insensitive with paraffin embedded tissue sections.Supported by the Sigrid Jusélius Foundation and Finska Läkaresällskapet