The principles and advances of quantitative pathology

This article gives an overview of the quantitative pathologic techniques used today, with special emphasis on interactive morphometry and its application in the clinical setting. At present, stereologic calculations may be necessary, but in a diagnostic setting have only rarely proved to be essential. The reproducibility of the measurements is discussed in relation to the definition of the particles and staining methods. A number of technical factors that cause random errors are mentioned, such as quality of the slides, magnification, definition of the particles to be measured and measuring protocol. Large-scale experiments have revealed that the means of nuclear morphometric and certain stereologic features are reproducible, such as in the volume percentage of epithelium, the surface densities of glands and the mitotic activity index. The diagnostic applications of the standard deviation and shape factors of nuclear quantitative features require additional precautions, however. Having quantified cell and tissue features, multivariate analysis may result in a better discrimination of two or more groups under study. The quantitative pathologic examination of cells and tissues can provide important diagnostic and prognostic information. Quantitation in pathology is especially useful in so-called continuous lesions, in which interobserver and intraobserver disagreement is considerable. An important requirement of diagnostic morphometry is object selection by a skilled pathologist; the use of morphometry as a black box can result in dramatic errors. The criteria used for a morphometric classification rule that can be used for clinical applications are summarized. Quality control of the whole measuring system is essential. Application of these techniques for more than six years in diagnostic pathology has repeatedly corrected previous qualitative diagnoses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular diagnostic cytopathology: definitions, scope and clinical utility

Molecular diagnosis is the application of molecular biology techniques and knowledge of the molecular mechanisms of disease to diagnosis, prognostication and treatment of diseases. Although it is not widely used in routine molecular cytological practice, some examples are presented here of the application of molecular techniques to the routine cytopathological diagnosis of solid tumours and lymphoreticular malignancies. The term 'molecular diagnostic cytopathology' is proposed to define the application of molecular diagnosis to cytopathology, and the challenges of the introduction of molecular diagnosis into routine diagnostic histopathology and cytopathology are discussed. Finally, the importance of a combined morphological, immunophenotypic and molecular approach to maintain the diagnostic pathologist at the heart of the clinical decision-making process is emphasized.     

Morphometric analysis of the degree of aggregation of follicular cells in the diagnosis of malignant thyroid tumors

OBJECTIVE: To develop a system of decision criteria based on a set of features of the degree of thyrocyte aggregation with assessment of their weighting coefficient in the diagnosis of malignant and benign thyroid pathology. STUDY DESIGN: Autopsy, operative and aspiration biopsy material were the object of the study. Light microscopic morphometry was performed with semiautomated computer analysis of images. RESULTS: Of all the sets of features of the degree of thyrocyte aggregation, 10 were chosen that provided the least overlap between the groups of malignant and benign pathology. Boundary values of these features to discriminate between malignant and benign pathology were assembled into a set of diagnostic decision criteria for thyroid carcinoma diagnosis. Based on results from trials, an equation for the calculation of a weighting coefficient for each feature was derived. A diagnostic index was determined by the sum of values of weighting coefficients of the features within the range of values listed in the set of diagnostic decision criteria. CONCLUSION: A set of diagnostic decision criteria was developed for thyroid carcinoma diagnosis that is based on morphometric features reflecting the regularities of changes in the structure of thyrocyte aggregates with malignant and benign pathology. Weighting coefficients of features were assessed, making it possible to perform a probabilistic carcinoma diagnosis.     

Application of morphometry in tumor pathology

The application of morphometry in tumor pathology is discussed, e.g., its use in studying the biology of tumors, in creating tumor classification(s), in creating methods for the identification of a tumor in the diagnostic context, and in characterizing diagnostic histopathology in absolute terms. In traditional subjective diagnostic histopathology, reproducibility can be defined satisfactorily, but the definition of accuracy is ambiguous; in morphometric histopathology, a satisfactory definition is found for both concepts but it may be difficult to separate them in practice. Morphometric histopathology can study parameters measured from sections or parameters derived from the primary measurements through calculations. In the histopathology of tumors, the following parameters have turned out to be specially valuable: densitometric measurements of nuclei, nuclear area, perimeter and form factors, nucleolar parameters, the number of mitotic cells per area, the cellularity, the volume fraction of the epithelium, and parameters associated with the fraction of tumor tissue in the sample. The standard deviation or other moments of the distribution of these measurements can be more relevant than the mean values of the results. This indicates that more attention should be given to sampling rules, which are important in defining the efficiency of the methods. For rational application of morphometric methods, it is very important to make a distinction between group morphometry and diagnostic morphometry. The latter engenders numerous sources of variation (variation in section thickness, variation in tissue processing, variation in the techniques of measurement, interobserver variation, interlaboratory variation, variation due to subjective interpretation, etc.), which are usually better controlled in group morphometry. The influence on morphometric parameters of variation in section thickness and tissue shrinkage during processing are discussed.     

Stereology and morphometry in histopathology. Principles of application

The role of stereology and morphometry (including image analysis) in histopathology is considered. Diagnostic histopathology has its own character, which should be considered when quantitative methods are applied. Three different types of studies in histopathology can be distinguished: prospective, retrospective and routine diagnostic studies. The variation in these studies is due to differences in the methods and differences in how the methods are applied. The overall variation is smallest in prospective studies and largest in routine diagnostic studies. Statistical classification of the overall variation does not cover all relevant aspects of the situation in which we study the sample from one individual patient. In that situation, we do not get support from the samples of other patients. In fact, we should speak about group morphometry (statistical morphometry) when samples from several patients are studied and diagnostic morphometry when the sample from one individual patient is under investigation. In the former case, simple morphometric methods may be applicable (the researcher is interested in mean values). In the latter case, one would often like to analyze the sample in more detail. This is why the diagnostic situation may benefit from computerized image analysis, which allows collection of large amounts of data in a short time. On the other hand, the great variation between the pathologic and the normal can allow the use of simple methods. The above basic principles need be considered before morphometric and stereologic methods are applied. These methods are simple in practice and they should be included in the training of all pathologists.     

Risk biomarker assessment for breast cancer progression: replication precision of nuclear morphometry.

Nuclear morphometry is a method for quantitative measurement of histopathologic changes in the appearance of stained cell nuclei. Numerous studies have indicated that these assessments may provide clinically relevant information related to the degree of progression and malignant potential of breast neoplasia. Nuclear features are derived from computerized analysis of digitized microscope images, and a quantitative Feulgen stain for DNA was used. Features analyzed included: (1) DNA content; (2) nuclear size and shape; and (3) texture features, describing spatial features of chromatin distribution. In this study replicated measurements are described on a series of 54 breast carcinoma specimens of differing pathologic grades. Duplicate measurements were performed using two serial sections, which were processed and analyzed separately. The value of a single feature measurement, the nuclear area profile, was shown to be the strongest indicator of progression. A quantitative nuclear grade was derived and shown to be strongly correlated with not only the pathologic nuclear grade, but also with tubule formation, mitotic grade, and with the overall histopathologic grade. Analysis of replication precision showed that the standard methods of the histopathology laboratory, if practiced in a uniform manner, are sufficient to ensure reproducibility of these assessments. We argue that nuclear morphometry provides a standardized and reproducible framework for quantitative pathologic assessments.     

The Papanicolaou-Traut method of cancer diagnosis; its use as a routine pathologic laboratory procedure

In a series of 450 examinations by the smear technique in the pathologic laboratory of a small private hospital, the total diagnostic error on all smears was 6.6 per cent. The error on vaginal smears was 4.7 per cent. These figures include false positives, false negatives, and all those smears classified as suspicious even though positive or negative diagnoses were made on subsequent examinations. The Papanicolaou-Traut method of cancer diagnosis can easily be made one of the routine pathologic procedures in the small hospital laboratory. Technicians with little previous cytologic experience can be trained to screen vaginal and other smears accurately after a short training period. This cytologic method is proving to be of value in the early detection of some neoplasms, and if its application is to be extended, the practicing pathologist should add the test to his diagnostic routine.     

THE PAPANICOLAOU-TRAUT METHOD OF CANCER DIAGNOSIS,Its Use as a Routine Pathologic Laboratory Procedure

In a series of 450 examinations by the smear technique in the pathologic laboratory of a small private hospital, the total diagnostic error on all smears was 6.6 per cent. The error on vaginal smears was 4.7 per cent. These figures include false positives, false negatives, and all those smears classified as suspicious even though positive or negative diagnoses were made on subsequent examinations.The Papanicolaou-Traut method of cancer diagnosis can easily be made one of the routine pathologic procedures in the small hospital laboratory. Technicians with little previous cytologic experience can be trained to screen vaginal and other smears accurately after a short training period. This cytologic method is proving to be of value in the early detection of some neoplasms, and if its application is to be extended, the practicing pathologist should add the test to his diagnostic routine.     

Basic aspects of and recent developments in telepathology in Europe, with specific emphasis on quality assurance

Telepathology is the diagnostic work of a pathologist from a distance and includes all specific fields of diagnostic pathology, such as frozen section services, expert consultation, cytometric and histometric measurement, and continuous education. For about 15 years experience has been collected at several universities in the United States and Europe based upon analog telephone lines (9.2 kbaud), digitized lines (ISDN, 64 kbaud), broad band connections (1.5 Mbaud) and the World Wide Web (28 kbaud). Potential use can be expected in the application of telepresentation, remote slide preparation, remote central diagnostics and telediscussion. The transfer of still images is well developed; that of live images is used in only a few institutions for frozen section services. The image quality and spatial resolution as well as the transfer speed are sufficient for expert consultations, morphometric measurements, quality assurance and education. All applications focus on discontinuous work flow. Although the European Community focuses on user needs and standardization aspects of telepathology by sponsoring a widespread telepathology project (Europath), implementation of telepathology into routine application in the continuous work flow has still to be developed. The technical equipment has still to be adjusted to the labor flow charts in routine pathologic diagnostic procedures. Telepathology seems to be the appropriate technique to offer both improvement in diagnostic quality and inclusion of the "control institution" into diagnostic responsibility.     

Standardization of nuclear morphometry-based biomarkers

Nuclear morphometry is used to address subtleties of carcinogenesis; it has been proposed for evaluating chemoprevention. An important issue for morphometry concerns control for extraneous sources of variation: fixation, slide cutting and staining. A common strategy has been to standardize the morphometric measures. Morphometric variables--such features as mean nuclear size and staining intensity--are often combined into multivariate indices. In this paper, we consider these variables one by one; any index is to a significant degree dependent on the individual indicators. This paper considers the extent to which statistical adjustment adds to the informational utility of individual indicators. We consider 14 features of 934 prostatic nuclei diagnosed by a single pathologist (Rodolfo Montironi) within a region of either normal tissue or high-grade prostatic intraepithelial neoplasia (HGPIN). HGPIN, a precursor to prostate cancer (PC), has been suggested as a target for PC chemoprevention. We consider a range of adjustment methods: transforming variables into deviations from means or from expected values generated by regression analysis. Our major test of standardization utility is the ability of the variables to deemphasize interindividual differences within diagnostic categories but to distinguish between diagnostic categories.     

Morphometry in surgical pathology

The potential role of morphometry in surgical pathology is discussed. Specific areas in which morphometry could be helpful are in (1) identifying malignant cells in lesions that are largely composed of benign-appearing cells (e.g., follicular thyroid neoplasms), (2) defining reference points in apparent continua (e.g., in the progression from normal colon to adenoma to adenocarcinoma), (3) distinguishing between benign and malignant lesions with similar appearances (e.g., fibromatosis and fibrosarcoma of the soft tissue) and (4) distinguishing between similar-appearing types of malignant neoplasms (e.g., between small-cell carcinoma of the lung and small-cell lymphoma). Morphometric techniques are already being used in DNA ploidy determinations, which frequently bear prognostic information. The measurement of other nuclear and cellular parameters has been used for both diagnostic and prognostic ends; one example is the relation of nuclear roundness to metastatic potential in prostatic carcinomas. Morphometry is now being increasingly applied to histologic sections, as in the prognostic study of lesion thickness in malignant melanoma and the diagnostic study of glandular architecture in colonic adenoma. The use of morphometry can enhance the observation and interpretation of morphologic features, which, combined with the clinical data and the experience of the pathologist, can lead to greater accuracy and precision in surgical pathology diagnoses.     

The use of ROC curves in histopathologic decision making

The applicability of receiver operator characteristic (ROC) curves to histopathology is presented in three examples, along with a basic discussion of some properties of ROC curves. One major application is in the attempt to define stages in diseases that show a continuous spectrum of histologic patterns, in which the uncertainty of boundary points and the overlap of features makes such definition difficult. Construction and analysis of ROC curves may help to identify the features with the greatest utility, as, for example, in the grading of mucinous carcinomas of the ovary. ROC curves can also be used to assess diagnostic differences between histopathologists, whether they are using different criteria or the same criteria but with different weightings, as, for example, in cervical premalignancy or borderline ovarian tumors.     

Further evaluation of the practical applicability of nuclear morphometry for the prediction of the outcome of atypical endometrial hyperplasia

A previously developed morphometric classification rule has been shown to be successful in identifying approximately 30% of patients with atypical hyperplasia of the endometrium in whom the finding does not imply a progression to malignancy. The reproducibility of the nuclear classification rule was tested in blind, duplicate morphometric assessments by different technicians. The results showed a satisfactorily high degree of consistency and reproducibility, with only one of ten cases classified differently. In a second series of experiments, the nuclear classification rule was applied to samples from 101 nonmalignant cases (39 proliferative endometriums, 7 secretory endometriums, 55 mildly atypical hyperplasias), 8 markedly atypical hyperplasias and 43 malignant cases (20 well-differentiated adenocarcinomas and 23 moderately to poorly differentiated adenocarcinomas of the endometrium). Ideally, the rule should classify all nonhyperplastic and mildly hyperplastic cases as nonprogressive and all carcinomas as progressive; there were, however, a considerable number of false positives and false negatives based on application of the classification rule to these cases. Therefore, the sensitivity and specificity of nuclear morphometry using the classification rule developed for atypical hyperplasias is too low to allow its random application. This emphasizes the selective nature of diagnostic morphometry, in which the full diagnostic capacity of the pathologist must be used in selection of the proper cases to be studied.     

植物分类学在化石珊瑚藻(珊瑚藻目,红藻门)中的应用

最近有人认为将化石藻类的分类归入现生藻类分类单元有利于珊瑚藻作为古环境的标志,便于理解该类群的演化。然而,这样分类可能很难,因为并不是所有现生藻类分类特征都能在化石种中保存下来。Sporolithacea科的钙化部分(独立或者聚集的孢子囊群)的出现,可以把它们与这个类群的另一个现生科Corallinaceae区别开,这个科在生殖窠中产生孢子囊。节片的有无,丝间细胞的联系类型,生殖窠中孢子囊释放的数目都是用来划分Coral1inaceae科的亚科的标准,在化石样品中也可以用合适的条件进行观察。在大多数情况下,对现生珊瑚藻类属的划分特征可以在化石藻类中鉴别出来,但在几种现生珊瑚藻没有钙化的生殖结构或发育特征。因此,它们生殖结构无法与相应的化石藻类进行对比,也不能进行化石藻类的分类。近年来的趋势认为生殖结构和发育特征是对现生珊瑚藻进行分类的优先鉴定标准,然而,某些特征的稳定性在属的划分上仍然存在争论。在许多情况下,现生藻类的分类标准特征都不能在化石中保存,对古生物化石的分类标准的最佳选择是在化石藻类中选择辅助的,并且可以识别的其它鉴定特征,或者应用非正式的比现生藻类代表定义更宽的属名。     

Automated region of interest retrieval and classification using spectral analysis

Efficient use of whole slide imaging in pathology needs automated region of interest (ROI) retrieval and classification, through the use of image analysis and data sorting tools. One possible method for data sorting uses Spectral Analysis for Dimensionality Reduction. We present some interesting results in the field of histopathology and cytohematology. In histopathology, we developed a Computer-Aided Diagnosis system applied to low-resolution images representing the totality of histological breast tumour sections. The images can be digitized directly at low resolution or be obtained from sub-sampled high-resolution virtual slides. Spectral Analysis is used (1) for image segmentation (stroma, tumour epithelium), by determining a "distance" between all the images of the database, (2) for choosing representative images and characteristic patterns of each histological type in order to index them, and (3) for visualizing images or features similar to a sample provided by the pathologist. In cytohematology, we studied a blood smear virtual slide acquired through high resolution oil scanning and Spectral Analysis is used to sort selected nucleated blood cell classes so that the pathologist may easily focus on specific classes whose morphology could then be studied more carefully or which can be analyzed through complementary instruments, like Multispectral Imaging or Raman MicroSpectroscopy.     

Clinical applications of the enzyme labeled antibody method. Immunoperoxidase methods in diagnostic histopathology.

Histological criteria for the definition of disease entities have largely been established with light microscopy of conventionally stained and routinely processed tissue sections. More or less specific histochemical staining procedures and more recently enzyme-histochemical and quantitative histo- and cytochemical techniques in some cases provided additional criteria. In the last decade, however, the introduction of immunofluorescence and more recently the different immunoperoxidase methods have significantly influenced the scope of contemporary histopathology. Especially, the possibility to use immunoperoxidase methods on routinely processed tissue specimens has offered new dimensions in diagnostic pathology. These methods proved of particular importance for: 1) The development of new criteria for diagnosis, prognosis and treatment (e.g. immunological classification of lymphoma; plasmacell typing in intestinal inflammatory conditions; human chorionic gonadotropin and alpha-fetoprotein in germ cell tumors of the testis). 2) The possibility of etiological diagnosis (e.g. the recognition of hepatitis B viral antigens in liver biopsy specimens; histological typing of causative micro-organisms in inflammatory conditions). 3) The recognition of disease entities that were hitherto unrecognized (e.g. hyperplasia of parafolicular C-cells in the thyroid in multiple endocrine neoplasia syndromes; gastric G-cell hyperplasia as a variant of Zollinger-Ellisons syndrome). 4) Functional analysis of tissue components (e.g. hormone content of pituitary and pancreatic adenomas; cytoplasmic differentiation produces in "undifferentiated" tumors). It can be expected that immunoenzymehistochemistry will soon play a major role in routine diagnostic histopathology.     

Telecytologic diagnosis of breast fine needle aspiration biopsies. Intraobserver concordance

OBJECTIVE: To determine the intraobserver concordance between telecytologic and glass slide diagnosis of breast fine needle aspirates. STUDY DESIGN: Twenty-five cases, originally received in consultation, were each examined by three cytopathologists. An average of seven compressed digital images per case were presented, together with a brief clinical history, using the http protocol and an internet browser. RESULTS: Agreement between the telecytologic and glass slide diagnosis ranged from 80% to 96%. Nevertheless, two cases that had been unequivocally diagnosed as malignant based upon video images were considered to be benign by the same pathologist when reviewing the glass slides. Both diagnostic confidence and self-concordance were higher for one pathologist having significant previous video microscopy experience. CONCLUSION: Although intraobserver concordance between telecytologic and glass slide diagnoses of breast fine needle aspirates is high, refinement of existing criteria for diagnosis of malignancy, taking account of the particular limitations associated with telecytologic diagnosis, may be prudent prior to widespread use of telecytology for fine needle aspiration evaluation.     

Cytologic diagnosis of metastatic germ-cell tumors

The clinical, pathologic and cytologic findings were correlated in 86 cases of metastatic germ-cell tumors. Although the cytologic features of malignant germ-cell tumors are sufficiently characteristic to make specific cytologic diagnosis possible, the diagnostic accuracy can be augmented with cytochemical stains. It was found that due to recent advances in therapy, cytologic detection of metastases does not necessarily indicate a fatal outcome.