morphometry.pptx

Morphometry

1. ROLE OF MORPHOMETRY IN HISTOPATHOLOGY Presentor-dr sandhya MODERATOR-DR PRAVEEN SIR

2. INTRODUCTION Histopathology is crucial for diagnostic and therapeutic decisions in many diseases. The created classifications, however, have not always allowed explicit recognition of diagnostic categories. So, considerable intra- and interobserver variations are possible under diagnostic circumstances. Better reproducibility can be reached by applying traditional methods like morphometry as well as special stains, electron microscopy and immunohistochemistry

3. What is Morphometry? A body of methods for obtaining information about shape and size of a structure in terms of quantities such as: Volume Surface area Relative amounts of each component Orientation, Interconnections Distribution of substructures

4. Morphometric approach includes item classification and grading, point counting and intersection counting methods, and the use of various semiautomatic or automatic instruments. TYPES :1.Statistical morphometry 2.Diagnostic morphometry

5. In statistical morphometry morphometrical parameters are collected from several disease cases. Such data reinforced with prognostic follow ups -are the basis for disease classification. Diagnostic morphometry , on the other hand, tries to study the sample of one individual and give relevant data for diagnostic decisions

6. WHAT IS MAJOR CHALLENGE? To extract information about large 3D structures from microscopic measurements on thin 2D sections To do this Histomorphometry uses the “ DELESSE PRINCIPLE ”

7. “In a rock composed of number of minerals, the area occupied by a given mineral is propotional to the volume of mineral in the rock” Repeated determinations of the area fraction will yield an estimate of the volume fraction. The more determinations; the better the estimate

8. Histomorphometry When applied to biological tissue, which is examined microscopically, it is useful in correlating structure and function. Ex- gut villus surface area Arterial composition and elasticity Quantification of –Hyperplasia, dysplasia, hypertrophy Immunohistochemical or fluorescent markers-area or intensity

9. “Planimetry" -The measurements of surface areas and perimeters by tracing the boundaries. Planimetry on photomicrographs or projected images may be used to evaluate the size of the cells With the widely commercially available digitizing instruments the perimeter, area, longest and shortest axis and also other features of individual elements can be measured with an acceptable speed

10. "stereology “-technique for extracting quantitative information about a 3 dimensional material from measurements made on two dimensional planar sections of the material. In practice denotes point counting techniques like performing cell counts, estimating volume of lesions, infarct. The two methods therefore supplement each other may lead to "morphometry", the quantitative description of a structure.

11. Why morphometry in Pathology? The pathologist may be intrested in morphometry for checking an initiative and subjective evaluation of a diagnostic feature on the microscope section by making measurements He does this because he understands that other pathologists may possibly interpret his findings differently

12. USES To study biology of tumours Classification of tumours Diagnostic histopathology Reproducibility and accuracy

13. Quantification of data The commercially available equipment for quantitation can be divided as follows: Non-automatic - Point counting equipment Semi automatic - Graphic (digitizing) tablets Mechanical scanners a. scanning stages b. mirror scanners c. polygon scanners Automatic - Electron beam scanners e.g. cathode ray tubes (CRT) Electron image sensors a. TV tubes b. diode array scanners Flow cytometer

14. Point Counting Equipment This equipment is especially suitable for the assessment of stereological features such as volume percentages, surface densities, length densities and numerical densities.

15. Graphic Tablets In order to measure the area, circumference (= perimeter) etc. of individual elements such as nuclei, a graphic tablet is necessary. This instrumentation consists of three basic components: an electromagnetic sensitive plate a pen ( or cursor) which is electrically connected with a memory. Every time the cursor on the plate is activated (e.g. by a microswitch), the exact location of the pen is recorded according to its x and y coordinates. This information is then stored in the computer

16. Graphic tablets are especially suitable for the measurement of individual structures (nuclei, cells and other elements). On most of the commercially available graphic tablets the following features and parameters can be calculated per delineated structure: 1. Perimeter ( = circumference) 2. Area 3. Shape factor (= 4 n x area/perimeter ) 4. Projection on x-axis 5. Projection on y-axis 6. Longest axis 7. Shortest axis 8. Ratio of longest and shortest axis

17. HOW TO ESTIMATE AREA FRACTION? Planimetry Dot counting Square counting Pixel counting in a digital image

18. DOT COUNTING Nuclear area/cell area=number of dots in nuclei/number of dots in cell Absolute area of structure=number of dots in structure x area of dot square

19. Pixel counting

20. Counting of mitotic figures The following criteria for the diagnosis of mitotic figures instead of "pycnotic deformed nuclei" 1. Absence of nuclear membrane 2. Absence of clear zone in centre 3. Presence of hairy instead of triangular or spiky projections 4. Basophilia of surrounding cytoplasm instead of eosinophilia

21. The following measures must be taken when counting mitotic figures 1. Adequate sections should be used, lightly stained, 4-5 microns in thickness. 2. Adequate sampling: As there may be considerable differences from one place to another, at least 10 sections per borderline tumour , or one section for each centimetre of diameter of the tumour should be taken, whichever is greater.

22. 3. Counts should be started in the most active areas where the number of mitoses is highest. Once having started in a certain area, nine additional contiguous fields should be selected randomly. No attempts should be made to maximize the counting by selecting those areas with a higher number 4.Four sets of 10 (or 25 if required) high power fields should be counted and the maximum number in anyone set used. 5. Only definite mitotic figures should be counted, doubtful structures should be excluded.

23. DECISION TREES A decision tree consists of a combination of sequentially taken decisions, on the basis of the value of one or more features, in order to come to a diagnosis. Step by step, the investigator is asked to make a decision.

24. The morphometric features themselves are expressions of the biological behaviour of the tumour . In general pathology the nuclei of malignant cells are usually described as being "enlarged, often irregular,pleomorphic ” In the applications of morphometry in tumor diagnostics, the nuclear area (related to size), the nuclear perimeter (related to size and shape) and the nuclear horizontal and vertical axes (also related to size and shape) are often of importance.

25. Pattern recognition

26. Sometimes the nuclear area has been used as a discriminating parameter in the morphometric applications, a phenomenon familiar to the diagnostic pathologist. Simple calculations such as the ratio of the nuclear axes are related to the shape of the nucleus.

27. BIOLOGICAL FEATURE MORPHOLOGICAL FEATURE MORHOMETRICAL FEATURE Evidence of rapid growth Mitosis numerous and often abnormal Mitotic activity index increased Nuclei enlarged and pleomorphic Mean and s.d of nuclear area Perimeter Longest and shortest axis increased Nucleoli usually large Mean of nucleolar area increased. Localization of nucleoli Cytoplasmic basophilia Haemorrhage and necrosis Area of cytoplasm increased n/c ratio increased Volume percentage of necrosis increased

28. MORE DIFFICULT MEASUREMENTS Length seminiferous tubules Surface area Alveoli, gut villi Counting Cells, nuclei

29. Endometrium: Hyperplasia or Carcinoma Morphometry has been applied to endometrial diagnostic pathology . The disagreement in the diagnoses both within the same pathologist and between different pathologists in this particular area of pathology emphasizes the obvious need for objective criteria. Further grading in mild and marked atypical hyperplasia and well and moderately to poorly differentiated carcinoma is also possible with morphometry

30. Morphometry of endometrium Volume % stroma -43.7% Volume % epithelium -50% Volume % lumen -6.2% Volume % glands-56.2% Outer surface density glands-6.6mm2 Inner surface density glands -6.6mm2

31. For discrimination of hyperplasia and carcinoma, measurement and calculation of the volume percentage epithelium and the inner luminar ) surface of the glands with stereology is sufficient

32. BORDERLINE VS MALIGNANT-OVARY The most powerful morphometric discriminators are the mitotic activity index and the volume percentage epithelium.

33. Mesothelial Cells: Morphometric Distinction Between Benign and Malignant in Pleural Fluid The nuclear and cytoplasmic areas of mesothelial cells are measured with a graphic tablet 1000x magnification. From each measured cell population the following six parameters are calculated: mean nuclear area standard deviation nuclear area, mean cytoplasmic area, standard deviation cytoplasmic area, mean N/C area ratio standard deviation N/C area ratio.

34. Mean nuclear area and mean cytoplasmic area with their standard deviation show the most significant differences. In the mesothelioma group the mean area and the mean cytoplasmic area are greater than in the group with reactive mesothelial cells.

35. Scattergram of reactive mesothelial cells (open circles), malignant mesothelioma cells (stars) and carcinoma cases (squares) with the standard deviation and mean of the nuclear area.

36. NEXTGENERATION MORPHOMETRY

37. FLASH Framework for large-scale histomorphometry (FLASH) performing deep learning based semantic segmentation and subsequent large-scale extraction of interpretable, quantitative, morphometric features .

38. FLASH consists of an automated three-step approach: ( i ) a CNN that automatically segments kidney tissue on a WSI discarding all non kidney tissues (e.g., adipose or muscle tissue), (ii) another CNN that segments histological structures of the kidney tissue segmented by the first CNN and (iii) hand-crafted feature extraction for segmented structures . The framework is applicable to the whole morphological spectrum of non-neoplastic kidney

39. COMPUTERZED HISTOMORPHOMETRY It is a method used to quantitatively analyse microscopic images of tissue samples 1.Sample preparation 2.slide scanning -slides are scanned by whole slide scanner to create high-resolution digital images. 3. Image Acquisition : The digital images are imported into a computerized histomorphometry software program.

40. 4.Calibration: The software is calibrated to ensure accurate measurement by setting scale factors based on known dimensions in the images. 5. Image Preprocessing : Preprocessing steps may include color correction, noise reduction, and image enhancement to improve the quality of the images. 6.Region of Interest (ROI) Selection : The user defines the regions of interest within the images where histomorphometric measurements will be performed.

41. 7.Segmentation : Segmentation involves distinguishing between different tissue components, such as nuclei, cytoplasm, and extracellular matrix. This can be done manually or using automated algorithms.

42. 8.Measurement : Various histomorphometric parameters are measured, including area, length, density, and shape characteristics of the tissue components within the ROI 9. Data Analysis : The software calculates and compiles the quantitative data based on the measurements, often generating statistics and graphical representations. 10.Interpretation : Researchers interpret the results in the context of their specific research questions, looking for patterns or differences in tissue morphology.

43. CONCLUSION Histomorphometry is useful for counting and measuring clearly defined structures Limited by a lack of “Intelligent” soft ware Extremely difficult to better the human eye-brain combination for pattern recognition and diagnosis. For Histopathologists, may be valuable for quantifying prognosis Measuring ratio or distribution of different tumor markers

44. REFERENCES 1. Hölscher DL,et al. Next-Generation Morphometry for pathomics -data mining in histopathology. Nat Commun . 2023 Jan 28;14(1). 2.Kalhan S, Garg S, Satarkar RN, et al.Correlation of Nuclear Morphometry with Clinicopathologic Parameters in Malignant Breast Aspirates South Asian J Cancer 2022;11(1). 3.Baak.j.Oort.A manual of morphometry in diagnostic pathology.1 st ed. springer;1983 3. Collan Y. Morphometry in pathology: another look at diagnostic histopathology. Pathol Res Pract . 1984 Nov;179(2): 4. Collan Y, Torkkeli et al. Application of morphometry in tumor pathology. Anal Quant Cytol Histol . 1987 May;9(2).