Gross Examination, Selection, Collection and Fixation of Specimen

Histopathology

1. Gross Examination, Selection, Collection and Fixation of Specimen Dr. Sohaib Aslam PhD Pathology Ass. Professor Dr Ghulam Abbas PhD Poultry Nutrition Ass . Professor Riphah college of Veterinary Sciences, Lahore

2. 2 1 Specimen Identification Grossing Collection Labeling 2 3 4 Fixation 5 Outline

3. The laboratory diagnosis of an infectious disease begins collection of a clinical specimen for examination or processing in the laboratory Rule: right one, collected at the right time, transported in the right way to the right laboratory. Proper collection first step accurate laboratory Dxs . Collection and transportation of specimens two important aspects. b/4 antimicrobial agents. Prevention of contamination of the specimen

4. Identification and Labelling Specimen Identification Animal/patient information History Description of site of origin. Labeling Give each specimen a number

5. Gross Examination In order to establish a diagnosis and to select relevant portions for microscopic examination Diagnoses on the basis of gross examination 90 % of specimens In the remaining 10 % the skilled pathologist can be close to the diagnosis Two mandatory prerequisites before examining Knowledge of the clinical history Thorough knowledge of the anatomy of the organ

6. Principles of macroscopic assessment or gross examination of a specimen or lesion Location of the lesion Size, weight, length Shape and architecture Color and consistency Describe cut surfaces and identify pathological changes

7. Selection and Collection of Specimen A basic rule is that the fewer sections are taken, the fewer slides will need to be reviewed. Without delay after death/sampling & fix Selected tissue should contain portion of lesion Take youngest lesion from periphery Take specimen from more than one areas of affected organs Cut must be made quickly, sharply and accurately Optimum thickness 5-6mm No pitching, squeezing, bending or crushing Wash blood with normal saline

8. Gross Specimen Photography Acquiring Gross Pathology Skills See as many gross specimens as possible Specifically study the specimen for the features already discussed Correlate the microscopic findings in each section with the gross findings of the specific area from which the section(s) have come.

9. Grossing of Specimen Cutting up specimens  is known as  grossing Grossing is an art Sectioning the tissue to be processed for diagnosis Cut the organ at interval of 1 cm thickness Histopathological sections ~5-6mm Put in metal cassettes/capsules and then in fixative Small specimen can be wrapped in tissue paper and then in cassette & fixed

10. Instruments Used in Grossing

11. Handling of Specimen Proper specimen handling from the animal/patient to the time a completed slide Handling in such a way that specimens deemed acceptable Identification of the patient sample (labeling) Specimen container to be used (Wide-mouthed screw-capped, leaked proof) Type and volume of fixation Transport packing, temperature and method Write “Biological Material”

12. Wide mouth screw-capped bottle

13. Fixation of Tissues The major objective to maintain clear and consistent morphological features as living stat In order to visualize the microanatomy of tissues, Stained sections of tissue must maintain the microscopic relationships among cells, cellular components (e.g. the cytoplasm and nuclei) Extracellular material with little disruption of the organization of the tissue Maintaining the tissue's local chemical composition. Prevent drying of tissue Each fixative has some disadvantages, like molecular loss, swelling, shrinkage, hardening of tissues and color verification

14. Conti.. The fixative acts to 'fix at a point in time' by minimizing the loss or enzymatic destruction of cellular and extracellular molecules, maintaining macromolecular structures and protecting tissues from destruction by Microorganisms Long-term storage Infectious agents Prevent the micro-architecture by activity of catabolic enzymes The most important characteristic of a fixative is to support high quality and consistent staining with hematoxylin and eosin (H&E)

15. Ideal Fixatives Prevent autolysis & bacterial decomposition Preserve tissue in their natural state & fix all components Make cellular components insoluble in liquids encountered in tissue processing Preserve tissue volume Avoid excessive hardness of fixed tissue Enhance staining of tissues Non-toxic & non-allergic

16. Types of Fixatives Fixatives can be classified in different ways depending on their mechanism of action. In the most general terms, there are physical and chemical methods of fixation. Physical fixation processes includes augmenting a fixative with heat or microwaving, Cryopreservation is another physical method The chemical fixatives can be classified a few ways Additives that form cross- links e.g. Formaldehyde, zinc sulfate, glutaraldehyde, picric acid and mercuric chloride. Non-additive those that denature, most commonly accomplished by dehydration e.g. methyl and ethyl alcohol

17. Classification of Fixatives A. Tissue fixatives a. Buffered formalin b. Buffered gluteraldehyde c. Zenker’s formal saline d. Bowen’s fluid B. Cytological fixatives a. Ethanol b. Methanol c. Ether C. Histochemical fixatives a. Formal saline b. Cold acetone c. Absolute alcohol

18. Factors Influencing Fixation Factors influencing fixation Recommendations Possible Negative effects Specimen dimensions 3 mm tissue sections are ideal 4 mm maximal thickness Thicker sections result in incomplete fixation Time of fixation Varies by fixative: Formalin—requires 6–8 h Bouin’s —not more than 18 h B-5 2–4 h then transfer to formalin Under-fixation can result in tissue distortion and poor staining; over-fixation with some fixatives (alcohols, B-5) can make tissue brittle or result in loss of antigenicity Penetration rate Depends on the diffusion characteristics of each fixative: Formalin penetrates at about 1 mm/h Fascia and capsules are naturally occurring physical barriers to fixatives and can dramatically decrease penetration. They must be incised prior to fixation Temperature Room temperature is ideal for the majority of tissue fixation and up to 45 °C during processing Heat increases the rate of fixation but also speeds up autolysis, which can result in poor morphology and staining

19. Factors influencing fixation Recommendations Possible Negative effects Volume ratio Generally accepted as 15–20:1 fixative to tissue ratio The concentration of active reagent in fixative diminishes as the chemical reaction of fixation occurs. If depleted, fixation will cease no matter how long tissue remains in the fixative pH and buffers Breakdown of formaldehyde results in formic acid which decreases pH. Buffers help avoid this by maintaining pH between 6.8 and 7.2 Formic acid reacts with hemoglobin to produce formalin pigment that deposits in tissue and can be misinterpreted as microorganisms or other pigments (melanin, iron); this can be removed from tissues in the staining process by short immersion of slides in Lugol iodine Osmolality Do not place tissue in water or leave in saline for excessive periods of time Cell lysis can occur if placed in hypotonic solution If immediate fixation is not possible, refrigerate, place on saline soaked gauze, or immerse in isotonic saline for a short period Cell lysis can occur if placed in hypotonic solution

20. Characteristics of an Ideal Fixative Useful for a wide variety of tissues. Preserve small and large specimens Penetrate and fix tissues rapidly Have a shelf life of at least one-year Compatible with automated tissue processors Readily disposable or recyclable Support long-term tissue storage Giving excellent microtomy of paraffin blocks Cost effective, non-toxic and non-allergic The most important is to support high quality and consistent staining with hematoxylin and eosin (H&E)

21. Common Fixatives: Uses and Limitations Fixative Primary/recommended use Limitations Alcohol Routine cytology Cases where gout is suspected Fixation for frozen sections, smears, and touch preps Methanol and ethanol cause cell shrinkage and will make tissue brittle specimens if over fixed Alcoholic formalin Completion fixation with incompletely fixed tissue; primary fixative for fatty specimens (allows for easier detection of lymph) Acidic pH can allow for formation of formalin pigment precipitates B-5 Hematopoietic and lymphoid tissue Sections require removal of mercury pigment prior to staining; tissue cannot be stored in this; low molecular weight or no extractable nucleic acid Bouin’s Gastrointestinal and genitourinary tissue Slowly removes small calcium and iron deposits; lysis of erythrocytes; low molecular weight or no extractable nucleic acid

22. Fixative Primary/recommended use Limitations Decalcifying solution (acid based) Large bone sections where future molecular testing is not required Poor staining with low molecular weight or no extractable nucleic acid. Prolonged immersion can completely dissolve specimen Formalin Routine processing Dissolves uric acid crystals; can dissolve breast microcalcifications if fixed >24 h prior to processing; reduced high molecular weight nucleic acids with time. Unbuffered formalin can allow for formation of formalin pigment precipitates Michel transport medium Renal biopsy transport Requires tissue to be washed with PBS Cases requiring immunofluorescence prior to processing Zenker’s Bone marrow biopsies Poor antigen preservation for IHC; slow penetration; contains mercury; lyses red blood cells Common Fixatives: Uses and Limitations

23. Neutral Buffered Formalin 10% Neutral buffered formalin (NBF) mostly used 40% formaldehyde gas in 100 w/v of distilled water= 100% formalin 10 mL of 100% formalin + 90 mL distilled water = 10% formalin 10 mL of 100% formalin + 90 mL distilled water + excessive Mg or Ca carbonates = 10% NBF formalin Mechanism of Action It forms cross links between amino acids of proteins nearby making them insoluble Make 4mm thick tissue fix in 8 h Advantages Rapid penetration, easy availability and cheap Does not over-harden the tissue, easily fixes lipids and carbohydrates Support the acid-base dyes

24. Disadvantages: • Irritant to the nose, eyes and mucous membranes • Formation of precipitate of paraformaldehyde (which can be prevented by adding 11- 16% methanol) • Formation of black formalin pigment (acid formaldehyde hematin