introduction to pathology, tissue processing. Histopathology and cytopathology techniques

introduction to pathology, tissue processing. Histopathology and cytopathology techniques

1. Item no. 01 Introduction. Preservation and transportation of specimen and tissue processing Dr. A K M Maruf Raza Associate Professor of Pathology

2. Introduction to Pathology Pathology is the study of the: structural, biochemical, and functional changes in cells, tissues and organs that underlie disease.

3. Pathology is divided into: General pathology : General pathology is concerned with the common reactions of cells and tissues to injurious stimuli. Systemic pathology : Concerned with the alterations and underlying mechanisms in organ specific diseases

4. The four aspects of a disease process : Etiology or Cause Pathogenesis Morphologic change Functional Derangements and Clinical Manifestations

5. Etiology or Cause Etiology or Cause can all be grouped into two classes: Genetic (e.g., inherited mutations and disease-associated gene variants, or polymorphisms). Acquired (e.g., infectious, nutritional, chemical, physical).

6. Pathogenesis Pathogenesis refers to: The cellular, biochemical and molecular events that occurs in cells or tissues to an injurious agent.

7. Morphologic change Morphologic changes refer to: The structural changes in cells or tissues that are characteristic of a disease process.

8. Functional Derangements and Clinical Manifestations The end results of etiology, pathogenesis and structural changes are: i . functional abnormalities leading to the clinical manifestations (symptoms and signs) of disease. ii. clinical course and outcome.

9. Histopathology Histopathology: Microscopic examination of stained tissue sections for diagnosis of disease is known as histopathology.

10. Biopsy Biopsy: It is the process of removal of tissue from the living body for the diagnosis of disease . Biopsy is done for : Confirmation of clinical diagnosis. Exclusion of differential diagnosis. To assess excision margin clearance in case of malignancy. Grading of malignancy. Staging of malignancy . Assessment of prognosis.  

11. Procedure in Histopathology/ Tissue processing in Histopathology

12. Procedures in Histopathology Tissue taken from the body as Biopsy by the Surgeon. The tissue is labeled properly giving details like name, age and sex in a requisition form, identity of the tissue, provisional diagnosis and other investigation findings.

13. Procedures in Histopathology 3. Tissue then put in a container with 10% formalin and send to the Pathology department for Histopathological examination. 4. The container containing tissue must be properly labeled and related clinical information should be given in a requisition form.

14. Procedures in Histopathology 5. In pathology department, tissue is registered in register book and the tissue sample is transfer to the Gross examination table . 6. The histopathologist examines the whole tissue, gives description of it, mentioning any abnormalities in the tissue and takes tissue blocks of 2-3 millimeters thickness for processing of tissue.

15. Procedures in Histopathology 7. Then the tissue block is taken for Tissue Processing and staining. The basic steps of tissue processing includes: 1. Fixation by 10% formalin. 2. Dehydration by Ethanol. 3. Clearing by Xylene . 4. Tissue Embedding by paraffin. 5. Tissue sectioning by Microtome (3 to 6 μ m thickness). 6. Staining ( Hematoxillin and Eosine ). 7. Cover-slip /Mounting of slides.

16. Fixative Routine fixative : 10% formalin. 10% formalin is the most commonly used routine fixative. Volume used Should be at least a 10:1 ratio of fixative to tissue. The commercially available formalin is 40% formaldehyde gas dissolved in water.

17. Fixative Other fixatives : (a) Zenkers fluid: It is preferred for fixing lymphnode , liver, spleen and bone-marrow. (b) Bouin’s solution : For preservation of Testis tissue. (c) Carnoys fixative: For rapid fixation within 6 hours. (d) Clarke’s solution. For electron microscopy fixatives used are: Glutaraldehyde . For frozen section no fixative is needed.

18. Aim of fixation: Enzymatic digestive processes (autolysis) are stopped thus the normal tissue structure is preserved. To facilitate staining of tissue . Tissue become hard after fixation which helps in microtome sectioning . Aid in visual differentiation of structure of tissue.

19. Stains used in Histopathology slide Hematoxylin and eosin (H&E) stain: It is the most common stain used for histopathology. It is a combination of two stain , hematoxillin , eosin. Hematoxylin has affinity for the nucleic acids of the nucleus and stains the nucleus appearing blue/ Violet in slide. Eosin stains the cytoplasmic proteins of cells and appears pink to Red in slide.

20. Other Histologic Stains 1. Periodic acid–Schiff (PAS) stain ( Mucin , Basement membrane) 2. Reticulin stain 3. Masson trichrome stain. 4. Oil red O stain (For fat and Lipid) 5. Congo red stain ( For Amyloid )

21. Cytopathology Definition: Cytopathology is the study of cells of different parts of body under microscope to diagnose different disease. It is rapid, minimal invasive and inexpensive diagnostic procedure. But gold standard of tumour diagnosis is Histopathological examination.

22. Cytopathological Method/Sample 1. Aspiration cytology (FNAC). 2. Exfoliative cytology ( Paps Smear). 3. Brush Cytology (Bronchial brushing, Stomatch brushing). 4. Imprint Cytology. 5. Fluid Cytology for malignant cells (Pleural fluid, Ascitic fluid).

23. Aspiration cytology This is the analysis of cells taken by aspiration by a needle from an organ or a mass of a organ. This procedure is called Fine Needle Aspiration cytology (FNAC) . A needle is inserted into the area of the body to aspirate the material. FNAC sometimes done with the use of imaging (e.g. ultrasound guided or CT guided FNAC ).

24. Exfoliative cytology This is the analysis of cells that are shed from body surfaces. Examples include the lining cells of the uterine cervix, Sputum, cells of urinary bladder. The analysis of cells from the cervix is called cervical or Papanicolaou smear (Pap smear).

25. Fixatives used in Cytopathology 95% Ethyl Alcohol (Ethanol). Propanol . 100% Methanol.

26. Stains used in Cytopathology Papanicolaou Stain ( Paps stain). May- Grunwald - Giemsa (MGG) Stain. Haematoxylin and Eosin (H&E) stain.

27. Immunohistochemistry Immunohistochemistry is a special histopathological technique. It is the additional investigative procedure for routine histopathology. Immunohistochemistry used to detect antigens within tissues.

28. IHC help in accurate diagnosis of malignancies where routine histopathology fails. IHC can help determine the origin of the tumour . IHC also plays role in treatment and prognostic evaluation. Estrogen and progesterone receptors (ER, PR) in breast cancer. ER, PR positive breast tumour has better prognosis.

29. Frozen Section It is a specialized histopathological procedure in order to make a rapid diagnosis of a tumour mass during surgery. The procedure usually takes only few minutes and result is communicated to the surgeon over telephone. Frozen sections are performed with an instrument called cryostat (Temperature is -20 to -30 celsius ).

30. Frozen Section In frozen section tissue is send fresh without any fixative. Uses: Rapid diagnosis : To detect whether a breast nodule is benign or malignant to decide lumpectomy or mastectomy. To see excision margin whether it is free or invaded by tumor. Diagnosis of hirusprung disease in neonates and children.

31. Microtome Microtome is an instrument used in histopathology to make thin tissue sections for microscopic study. Types: Rotary microtome Sliding microtome Freezing/ cryo microtome By microtome tissue sections are made usually in 3-6 micron thickness.