Histopathology specimen processing

Specimen handling, fixation, grossing, and stainig

1. Histopathology specimen processing AZFAR NEYAZ, JUNIOR RESIDENT SGPGIMS, LUCKNOW

2. Specimen identification and labeling Grossing & Fixation Dehydration Clearing Impregnation Embedding Microtomy Staining and Mounting Outline

3. Specimen identification and labeling Tissue specimen received in the laboratory have a request form that lists the patient information, history & description of the site of origin. The specimen are accessioned by giving them a number that will identify each specimen for each patient

4. Information must be provided Patient Identification Identification of individual requesting examination Procedure date Adequate clinical history and physical examination Organs resected or biopsied Orientation if required. Intraoperative findings Prior surgery or pathologic diagnosis Prior treatment Mention if rapid diagnosis is required.

5. Complex series of chemical events to preserve the basic structure of the tissue in such a manner that subsequent examination may be made to determine the microanatomy and allow localisation of its various chemical constituents The purpose of fixation is to preserve tissues permanently in life-like state as possible Fixation of tissues

6. 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

7. Types of fixatives Physical methods: Heating, Microwaving, Freeze drying Chemical methods Coagulatant fixatives :(Precipitate and coagulate protein) Alcohols and acetone Picric acid and trichloro acetic acid Cross linking fixatives: Formaldehyde and glutaraldehyde Other aldehydes (chloral hydrate, glyoxal ) Metal salts (Mercuric and zinc chloride, osmium tetroxide ) Compount fixatives

8. 10% neutral buffered form(NBF) is most commonly used fixative It is prepared by mixing 40% formaldehyde gas in 100 w/v of distilled water. The resultant mixture is 100% formalin. Routinely, 10% formalin is used which is prepared by mixing 10ml of 100% formalin in 90ml of distilled water. Mechanism of action: It forms cross links between amino acids of proteins thereby making them insoluble. It fixes 4 mm thick tissue in 8 hours . The fixative should be 10 times more in volume then the specimen. Formalin

9. Advantages: Rapid penetration Easy availability & cheap Does not overharden the tissue Fixes lipids for frozen sections 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 ).

10. Other Fixatives Glutaraldehyde and Osmium Tetraoxide B5 fixative: stock solution: Mercuric chloride, sodium acetate & distilled water Add 2ml of formaldehyde to 20ml of stock solution Zenker's fluid Bouin’s Fluid

11. Decalcification It is the process of removal of the calcium salts from the specimen. The various agents used for decalcifying are: Nitric acid Hydrochloric acid Formic acid Picric acid Acetic acid Citric acid

12. Grossing Grossing of specimen is important stage in surgical pathology. It involves: Accurate naked eye description of intact specimen Correct method of sectioning Gross examination of cut surface Selection of proper tissue blocks for microscopy Instructions for embedding & block making. Histopathology specimens are a vital in patient care. They not only establish tissue diagnosis, but are crucial in clinical management & provide important prognostic data.

13. Grossing is an art A knowledge of what needs be taken for microscopic study is crucial for final diagnosis.

14. Large room, well illuminated and properly ventilated. Cutting board placed inside a metal box designed in such a fashion that all the fluids flow directly into the sink. Shelves for specimen container. Ready access to a sink with hot and cold water. Ready access to formalin Box of instruments, Box with cassettes, labels. Large formalin container, photographic facilities. Large table with sink for large specimens Central table for multiple purpose. Grossing room

15. Large scissors Dissecting scissors Enterotome Saw scalpel Instruments used in grossing

16. Forceps Ruler BLADE Cutting board GLOVES Instruments used in grossing

17. Inking Resection margins Embedding instructions Orientation Distinguish between samples Identify the cut surface

18. General principles of grossing Specimen identification Identify all the anatomical structures present Orientation markers should be identified Measurements: Length, breadth, height Weight-especially parenchymatous organ Examine the external surface. Cut all the organs at intervals of 1cm thickness Describe cut surface, identify pathologic process If suspected lesion is present, measure, describe with colour & consistency. Surgical margins Histological sections

19. Number of fragments, aggregate dimension Greatest dimension of largest fragment Shape of the fragment Colour and consistency Should not be cut or inked All small biopsies must be supported within the cassette to prevent tissue loss during processing. All fragments are submitted. Small fragments may be dipped in eosin to make them more visible. Small biopsies

20. Lymph node 1. If the lymph node is received in the fresh state, cut 2–3mm slices perpendicular to the long axis- Take a small portion for culture Make imprints smears from the cut surface Submit tissue for flow cytometry , cytogenetics & molecular genetics Rest of tissue fix in formalin, and submit for histology. 2. If the specimen is received in formalin, submit representative sections. Description State whether node received fresh or fixe Size, number of node and condition of capsule Appearance of cut surface: color , nodularity , hemorrhage , necrosis Sections for histology One to three sections including capsule, depending on size of node

21. Cut parallel slices along the long axis through hilum while the specimen is fresh and examine each slice carefully for focal lesions Description Weight and dimensions Hilum : Nature of vessels, presence of lymph nodes, presence of accessory spleens Capsule: color , thickness, focal changes, adhesions, lacerations Cut surface: color , consistency, bulging, fibrous trabeculae , nodules or masses, diffuse infiltration Spleen – splenectomy

22. Sections for histology Incidental splenectomy : one section, including capsule Traumatically ruptured spleen:1 section through tear & 1away from it Diseased spleen: at least 3 sections, 1 from hilum & two from capsule Suspected Lymphoma : All grossly abnormal/suspicious areas; if no gross abnormalities seen, four random pieces

23. Thymus gland – thymectomy Procedure Weigh the entire organ. Cut parallel slices Look carefully for lymph nodes around the thymus Description Weight and dimensions; both lobes identifiable? Relative amount of fat and thymic parenchyma Tumour characteristics: size, shape, external appearance Cut section:color , necrosis,hemorrhage , fibrous band, calcification Attached structures (pleura, pericardium, lung, lymph nodes)

24. Sections for histology : Tumor : 3 or more sections, at least two of which should include capsule Uninvolved thymus: 2 sections Other organs, if present (lung, lymph nodes)

25. <4 mm 4-8 mm Skin – excision for benign lesion Specimens upto 3mm Specimens upto 3-6mm Specimens more than 6mm

26. Skin – excision for malignant tumor Small specimens  – up to 5cm in greatest length

27. Thyroid gland– Thyroidectomy Procedure Weigh and measure the specimen Orient the specimen and cut parallel longitudinal slices 5 mm each Search for parathyroid glands in the surrounding fat Description Type of specimen: lobectomy , subtotal thyroidectomy , total thyroidectomy Weight, shape, size, color and consistency of specimen Cut surface: smooth or nodular if nodular: number, size & appearance of nodules(cystic, calcified, hemorrhagic, necrotic) Encapsulated or invasive, distance to line of resection

28. For diffuse/Inflammatory lesions Three sections from each lobe and one from isthmus

29. For multinodular thyroid glands One section of each nodule (up to five nodules), including rim & adjacent normal gland. More than one section for larger nodules

30. Solitary Encapsulated Lesion For a solitary encapsulated nodule measuring up to 5cm: Entire circumference Most of these sections should include the tumor capsule and adjacent thyroid tissue 1 additional section for each additional centimeter in diameter.

31. Grossly invasive carcinoma other than papillary Three sections of tumor Three of non- neoplastic gland One or more from line of resection

32. For papillary carcinoma The glands needs to be sampled extensively due to multicentricity of tumour . Minimum 5 bits to be taken from gland proper in addition to those from line of resection.

33. Parathyroid glands– Parathyroidectomy Removal of diseased parathyroid glands is usually complete, but in cases of hyperplasia a portion of one gland is retained Procedure Accurately weigh each gland on a delicate balance after removing the surrounding fat Accurately label each parathyroid gland as to site Description Weight, color , consistency, and external appearance of each gland Sections for histology All parathyroid tissue Except for markedly enlarged gland in which minimum of three sections should be taken

34. Adrenal gland– adrenalectomy Procedure : Ink the specimen Cut parallel sections at 5 mm intervals in the transverse plane Description : Size and weight External surface: smooth, bosselated , shape of gland preserved Cut surface: color , necrosis, hemorrhage , cystic changes, encapsulation, extension into the surrounding tissues Sections for histology : Tumor if present, trying to demonstrate relationship with normal gland, tumor capsule, and surrounding organs Non- neoplastic gland, some including adrenal vein

35. Esophagus – Esophagectomy Procedure Dissect the specimen in the fresh state Open longitudinally from one end to the other (opposite the tumor ) If a portion of the stomach is included, open along the greater curvature in continuity with the esophageal cut Dissect the periesophageal fat and look for lymph nodes Divide into three portions: adjacent, proximal & distal to the tumor Paint the surgical specimens with ink after the specimens are fixed

36. Description Length & circumference of specimen; proximal stomach included (indicate length along lesser & greater curvature) Tumor : size, appearance, circumferentially, depth of invasion; extension into stomach, distance from both lines of resection Mucosa: Appearance of non- neoplastic mucosa; recognizable esophageal mucosa distal to tumor evidence of Barrett esophagus , lumen dilated proximal to tumor Wall: thickened, Varices Stomach, if present: features of cardioesophageal junction and gastric mucosa Lymph nodes: no, size of largest; appear grossly involved by tumor

37. Sections for histology Tumor : 4 longitudinal sections,1 including a portion of non- neoplastic mucosa proximal to tumor & another portion distal to tumor Non- neoplastic mucosa: 2-3 transverse sections, at different distances from tumor edge, proximally and/or distally, depending on location of tumor Stomach, if present: two sections, one including gastroesophageal junction Proximal and distal line of resection Lymph nodes: Adjacent to tumor , Proximal to tumor , Distal to tumor

38. Section should always be taken perpendicular to the direction of mucosal folds

39. Stomach – Gastrectomy for tumor Procedure Open the specimen along the greater curvature (unless the lesion is in this location; if it is, open the specimen along the lesser curvature) Dissect the lymph node groups If a splenectomy is included, dissect the hilar lymph nodes, measure and weigh the spleen, Paint the surgical margins In general, take the sections perpendicular to the directions of the mucosal folds If a thorough mapping of the mucosal abnormalities is desired, the entire specimen can be submitted or the Swiss roll technique can be used

40. Description Type of resection(total/subtotal); length of curvatures & duodenal cuff Tumor characteristics: Location, size, shape, depth of invasion; presence of serosal involvement; blood vessel invasion; extension into duodenum; distance from both lines of resection Appearance of non- neoplastic mucosa Sections for histology Tumor : 4 sections through wall & including tumor border and adjacent mucosa Non- neoplastic mucosa: mid stomach, two sections PRM along lesser & greater curvature: two sections each DRM (along pylorus & duodenum): two sections Spleen and Pancreas, if present Lymph nodes

41. If mapping of mucosal abnormalities are desired such as in metaplasia , dysplasia a swiss roll technique can be used, where mucosa alone can be shaved off from underlying muscle layer and rolled up

42. Stomach – Gastrectomy for ulcer Description Type of resection; length of greater, lesser curvature & duodenal cuff Ulcer characteristics: location, size, depth, shape and color of edges; perforation at ulcer base; appearance of serosa . Appearance of uninvolved mucosa: atrophy, edema , hemorrhage Sections for histology Ulcer: at least four sections Lesser curvature: two sections cut from proximal margin of excision Greater curvature: two sections cut from proximal margin of excision Pylorus and duodenum: two sections, including distal line of resection Other lesions, if present Lymph nodes: up to three sections

43. Large bowel: Colectomy for nontumoral condition Procedure Sample lymph nodes & remove the mesentery while the specimen is fresh Open the bowel longitudinally and fix overnight in formalin Take the sections perpendicular to the direction of the mucosal folds

44. Description Part of bowel removed, length of specimen & amount of mesentery Mucosa: type of lesions, extent, ulceration (linear or transverse), depth, pseudopolyps , hemorrhage , fissures Wall: thickening (focal or diffuse), atrophy, fibrosis, necrosis Serosa : fibrin, pus, fibrosis, adherence of mesentery Diverticula : number, size, location in relation to teniae , content, evidence of inflammation, hemorrhage or perforation Sections for histology As many as necessary to sample abnormal areas Proximal and distal lines of resection in cases of colitis Appendix, if included in specimen

45. Large bowel – colectomy for tumor Sections for histology Tumor : at least three sections (extending through the entire wall) Representative section of subserosal connective tissue, fat, and blood vessel around tumor Other lesions of bowel Proximal and distal line of resection Bowel between tumor and distal line of resection (halfway or 5 cm, whichever suits the case) Appendix, if included in specimen Lymph nodes: Around tumor , Distal to tumor , Proximal to tumor In abdominoperineal resections: anorectal junction

46. LYMPH NODES ACCORDING TO TOPOGRAPHICAL SITES

47. Polypectomy Procedure Measure the diameter of the head & length of the stalk For polyps with a short stalk or no stalk, identify the surgical section and cut in half longitudinally. For polyps with a long stalk (≥1), cut a cross-section of the stalk near the surgical margin and then cut the polyp longitudinally. Description Dimensions of polyp; diameter of head and length of stalk Polyp sessile/ pedunculated , Ulcerated, surface: smooth/papillary Sections for histology One longitudinal section(including surgical margin in polyps with short stalk or no stalk)   One cross-section of base of stalk (in polyps with long stalk).  

48. Small bowel – excision Procedure Cut longitudinally through the antimesenteric border & fix overnight Description Length and diameter of specimen, Wall: thickness, abnormalities Mucosa: appearance; edema , hemorrhage,ulcerations , tumor (size, location, circumferential involvement, depth of invasion) Serosa : fibrosis, peritonitis, adhesions Lymph nodes: size and appearance Mesentery; mesenteric blood vessels Sections for histology Depends on pathology present In cases of infarct: several cross-sections of mesenteric vessels

49. Appendix Procedure Measure organ (length and greatest diameter) Divide specimen in two by cutting a cross-section 2 cm from tip Cut cross-sections of proximal fragment at 5 mm intervals Divide distal fragment in two by a longitudinal cut Description Length and greatest diameter External surface: fibrin, pus, hyperemia Perforation, condition of mesentery Wall: any localized lesions Mucosa: hyperemic , ulcerated Lumen: obliterated, dilated, Content: fecaliths , stones

50. Sections for histology Proximal 1/3 close to surgical margin: one cross-section. Mid one-third: one cross-section Distal one-third: one longitudinal section If tumor is present in the specimen, paint the surgical margin with India ink and take an additional section from it

51. Several types of mastectomy procedures Halsted type radical mastectomy Modified radical mastectomy (also known as extended simple mastectomy & total mastectomy) Simple mastectomy Subcutaneous mastectomy Tylectomy (lumpectomy, excisional biopsy)

52. Breast : Lumpectomy / Excisional biopsy Procedure : Measure the specimen and orient Section specimen: if specimen is ≤ 3cm, cut 3–4 mm slices; if it is larger, bisect specimen transversely, place cut surface down, and take sagittal blocks through superior and inferior portions Description : Dimensions and consistency of specimen Appearance of cut sections: fibrosis, cysts, calcification, tumor (size, color , borders, consistency, distance from surgical margins) Sections for histology : Small specimens: submit in their entirety (up to five cassettes) Larger specimens: thorough sampling should be done This include any grossly visible lesions & inked surgical margins

53. Breast : Wide local excision

54. Procedure & Description Side, type of mastectomy Orient specimen and take dimensions Structures included in specimen: skin, nipple, breast, major and minor pectoralis muscles, fascia, axillary tissue Features of external appearance:   Shape and color of skin Location and extent of skin changes Appearance of nipple and areola Location of lesions Features of cross-sections: Mass: Quadrant, depth beneath skin, size, shape, consistency, necrosis, hemorrhage , calcification, relation to skin, muscle, fascia Lymph node - No, size, locations of nodes with grossly evident tumor Mastectomy

55. Sections for histology Breast: Take three sections of tumor ; sample all lesions noted grossly and least one section from each quadrant Pectoralis major muscle (in radical mastectomies): take one section from any grossly abnormal area Nipple areolar complex- one section Lymph nodes : All identified nodes should be processed. Small nodes are submitted entirely; nodes >0.5cm sliced. If the axillary fat is grossly involved, a representative section should be taken. Label in the following order:    Radical mastectomy:   •  Low, Mid & High axillary lymph nodes . •    Interpectoral nodes Modified radical mastectomy:   •     Lower half    •     Upper half

56. Kidney – nephrectomy for nontumoral condition Procedure Measure and weigh the organ Cut the kidney sagittally , strip the capsule, and carefully open the pelvis, calyces, and ureter Take photographs & identify in one of them the sites of the sections to be taken If stones are present, submit for chemical analysis, if indicated

57. Description Weight and size of kidney Capsule: amount of pericapsular tissue, thickness, adherence External surface : smooth scars; cysts( no, size, location, content) Cortex: color , width; striations apparent and orderly Medulla: color , width; medullary rays apparent and orderly Pelvis: size; dilated blunting of calyces, Stones Ureter : diameter, length, evidence of dilation or stricture Renal artery and vein; appearance

58. Sections for histology Kidney: Three sections, each including cortex and medulla Pelvis: two sections Ureter

59. Kidney – Nephrectomy for tumor Procedure Cut the kidney sagittally , strip the capsule, and carefully open the pelvis, calyces, and ureter Description Weight and dimensions of specimen; length & diameter of ureter Tumor characteristics: size, shape, location, extent, homogenicity , necrosis, hemorrhage ; invasion of capsule, perirenal tissues, calyces, pelvis, and renal vein Uninvolved kidney: external surface, cortex, medulla; any additional focal lesions Pelvis: dilated blunting of calyces,Stones Presence, number, size, and appearance of perirenal lymph nodes

60. Sections for histology Tumor : RCC: minimum of 3 sections (including one with adjacent kidney); Pediatric tumors : minimum of one section/cm of tumor diameter; Carcinoma of renal pelvis: minimum of 3 sections with adjacent pelvis and/or renal parenchyma Kidney not involved by tumor : two sections Pelvis: one section in cases of RCC or pediatric tumors ; two sections in cases of carcinoma of renal pelvis Renal artery and vein- one section Ureter : one section in cases of RCC or pediatric tumors ; one section/cm of ureter resected (and any abnormal-looking areas) in cases of carcinoma of renal pelvis Lymph nodes, if present

61. Bladder – cystectomy Two options are available for the dissection:    Open with scissors in a Y shape through the anterior wall and fix overnight in formalin Fill with formalin, fix overnight and divide into anterior and posterior halves by first cutting the lateral bladder walls and then sectioning the prostate, beginning at the bladder neck and being careful to make the cut through the urethra. Description Size of bladder; length of ureters ; other organs present Tumor characteristics: size , location, extent of invasion, shape (papillary, ulcerated); multifocal lesions? Appearance of non- neoplastic mucosa; thickness of bladder wall away from tumor

62. Tumor : At least three sections, through bladder wall Bladder neck: one section Trigone : two sections Anterior wall: two sections Posterior wall: two sections Dome: two sections Any other abnormal-looking area in bladder mucosa Ureteral orifices, including intramural portion Ureteral proximal margins In males: prostate (two sections from each quadrant) and seminal vesicles (one section from each). Other organs present Perivesical lymph nodes, if any Sections for histology

63. Prostate gland:Transurethral resection(TUR) Procedure Examine all the fragments: Ca prostate is often yellow and/or hard; submit for histology chips with these gross characteristics Description Weight of specimen Size, shape, and color of chips Sections for histology All of specimen until four cassettes filled If an excess, one additional cassette for each additional 10 g of tissue If carcinoma is identified microscopically and then remainder of the tissue should be processed in its entirety, regardless of amount

64. Prostate gland – suprapubic prostatectomy for nodular hyperplasia Procedure Step section the specimen into 3 mm slices Examine each slice carefully for areas suspicious of carcinoma Description Weight of specimen Shape, color , and consistency Presence of hyperplastic nodules, cysts, calculi, areas suspicious of carcinoma Sections for histology Left lobe: three sections Right lobe: three sections Middle lobe: one section

65. Prostate gland: Radical prostatectomy for tumor Procedure Orient the specimen and paint the surgical margins with India ink Shave the vasa deferentia , bladder neck and apical margins Serially section the prostate at 2–3 mm intervals from apex to base Lay the individual slices sequentially and examine them carefully. Use the cross-section of the urethra as a landmark Take photographs of the slices & mark the site of the sections taken Description Weight and dimensions of specimen Organs present: Prostate, Urethra, s. vesicles, vas, lymph nodes Prostate: tumor (location in lobes, size, color , borders, capsular and periprostatic extension)   Urethra: patent? impinged by tumor ? Seminal vesicles: involved by tumor ?

66. Sections for histology Vasa deferentia margin Proximal (bladder neck) margin & distal (apical) margin Seminal vesicles: proximal, mid & distal portions from each side Prostate: The individual slices can be submitted in toto by cutting the slices to fit the standard cassette (right and left halves & anterior and posterior quadrants)

67. Penis – Penectomy Procedure : Introduce a catheter through the urethra and fix overnight at 4°C Paint the surgical margins (including the urethra) with India ink Cut longitudinally through the center : it should cut the urethra in two Description Type of operation: partial, total, scrotal skin, testicles, inguinal nodes Length and diameter of specimen Tumor : location in relations to glans , prepuce, skin, and urethra; size, color , borders, depth of invasion Glans penis & urethra: invaded by tumor ?. Sections for histology Tumor : three sections Glans penis and urethra Surgical margin (including urethra)

68. Testicle – orchidectomy Procedure Open the tunica vaginalis and cut the testicle sagittally Take photographs and identify the sites of the sections to be taken Cut serial slices of each testicles, perpendicular to the original section Cut the epididymis longitudinally throughout its entire length Make several cross-sections of the spermatic cord at several levels Description Weight and dimensions of testicle Length of spermatic cord Features of tumor : size, color ; consistency; homogeneity or lack of it; presence of cysts, necrosis, hemorrhage , bone, or cartilage; tumor extension to tunica albuginea , epididymis , cord, and other structures Features of non- neoplastic testicle: atrophy? fibrosis? Nodules? Features of rete testis and epididymis

69. Sections for histology Tumor : At least 3 sections or one section/cm of tumor , At least one of which should include some uninvolved testicle. Most of the sections should include tunica albuginea . Always submit sections from hemorrhagic or necrotic areas of tumor , as well as from solid or fleshy areas. Uninvolved testicle: two sections Epididymis Spermatic cord and surrounding soft tissue at a point about 1 cm from testicle: one cross-section Spermatic cord and surrounding soft tissue at line of resection: one cross-section

70. Uterus – cervical biopsy Procedure : Do not cut the specimen unless the individual pieces are ≥ 4 mm It is essential that all of the tissue received be processed Always carefully search the container and the underside of the lid Description : Number of pieces received, shape and color Measurement in aggregate Epithelial erosions? irregularity in epithelial thickness? Any evidence of tumors or cysts? Sections for histology : Submit the material in its entirety If specimens are received with a specific identification, label and submit them separately

71. Cervical cone Biopsy Procedure Specimen should be received intact with a suture identifying the 12 o’clock position Open the specimen by cutting longitudinally along12 o’clock position. Paint surgical margins with India ink Cut the entire cervix by making parallel sections, 2–3 mm apart, starting at the 12 o’clock position and moving clockwise. Sections should be taken in such a way that the epithelium is present in each section; Description : Size and shape of cone; complete cast of cervix or fragmented? Epithelium: color , presence of irregularities, erosions, masses (size, shape, location), cysts (size, content)

72. Sections for histology All of the tissue must be submitted (except for trimming of the stroma ) If the cone has been oriented to the 12 o’clock position, identify separately: Sections from 12 to 3 o’clock Sections from 3 to 6 o’clock Sections from 6 to 9 o’clock Sections from 9 to 12 o’clock

73. Uterus – hysterectomy Procedure: (For Non-malignant lesion) Measure and weigh the specimen Open it through both lateral walls, from the cervix to the uterine cornua Make a mark as to which half is anterior Make additional cuts through any large mass in the wall Make parallel sections through each half, about 1 cm apart Make several sections of the cervix along the endocervical canal Make at least one cross section of every myoma present, larger myomas need additional cuts If tubes and/or ovary accompanies the specimen, follow instructions for these organs

74. Description Type of hysterectomy: total, radical with salpingo-oophorectomy Shape of uterus: deformed, subserosal bulges Serosa : fibrous adhesions Wall: thickness, abnormalities Endometrium : appearance; thickness; polyps(size, shape); cysts Cervix: Ectoocervix , squamocolumnar junction, endocervical canal; Myomas : number, location; size; sessile / pedunculated,hemorrhage , necrosis, or calcification

75. Sections for histology : Corpus: at least two sections taken close to fundus and including endometrium , good portion of myometrium and, if thickness permits, serosa ; Additional sections from any grossly abnormal areas Myomas : at least one section per myoma , up to three; sections from any grossly abnormal area (e.g. soft, fleshy, necrotic, cystic) Cervix: one section from anterior half and one from posterior half Cervical or endometrial polyps: to be submitted in entirety unless extremely large

76. Hysterectomy For Malignant Tumours Description Tumor : Location, size, appearance, color , extent of endometrial extensions, presence of myometrial , serosal , parametrial (soft tissue), venous, cervical, or tubal extension Sections for histology Three sections, one of which should be through area of deepest invasion and be complete sections from surface of endometrium through serosa Two sections from non- neoplastic endometrium ; Soft tissue from left and eighth parametria If no obvious tumor present, sample entire endometrium by making parallel sections, 2–3 mm apart,

77. Hysterectomy for cervical carcinoma Amputate the cervix from the corpus about 2.5 cm above the external os Open the cervix with scissors through the endocervical canal at the 12 o’clock position Sections for histology Cervix: all tissue is submitted and identified separately from 1-12 o’clock Vaginal cuff (entire line of resection) Left soft tissue (for invasive cases only) Right soft tissue (for invasive cases only) Rest of uterus: Ovaries and tubes: Lymph nodes,

78. Ovary – oophorectomy Description Size and shape; weight, if enlarged Capsule: Thickened, adhesions, hemorrhage or rupture Cut section: character of cortex, medulla, and hilum ; cysts (size and content); corpus luteum , calcification, Hemorrhage Tumors : size; external appearance: smooth or papillary, solid or cystic,content of cystic masses; hemorrhage , necrosis, or calcification. Sections for histology : For incidental oophorectomies : one sagittal section of each entire ovary, For cysts: up to three sections of cyst wall (particularly from areas with papillary appearance) For tumors : 3 sections or one section/cm of tumor , whichever is greater and one section of non- neoplastic ovary, if identifiable

79. Fallopian tubes – salpingectomy Description Length and greatest diameter Serosa : fibrin? hemorrhage ? fibrous adhesions to other organs? Wall: abnormally thick? Ruptured? Mucosa: atrophic? hyperplastic ? appearance of fimbriated end Lumen: patent? dilated? content; diameter, if abnormally large Masses: size, appearance, invasion Cysts in paraovarian region: diameter, thickness of wall, content; sessile or pedunculated ? In cases of suspected ectopic pregnancy: embryo or placenta identified? amount of hemorrhage ; rupture?

80. Sections for histology For incidental tubes without gross abnormalities: Submit three cross-sections of each tube, taken from the proximal, mid, and distal portions, submitted in the same cassette

81. For tubes with suspected ectopic pregnancy: Submit any tissue with gross appearance of products of conception. If none is grossly identified, submit several sections from the wall in the area of hemorrhage as well as several from the intraluminal clot . If products of conception are not identified microscopically, submit additional sections For tubes with other lesions: As many as needed to adequately examine any abnormal areas. If tumor is present, at least three sections must be taken to include grossly uninvolved mucosa

82. Tissue processing Designed to remove all extractable water from tissue, replacing it with support medium that provide sufficient rigidity to enable sectioning of the tissue without damage or distortion Factors affecting rate of processing: Agitation Heat Viscosity Pressure Stage of tissue processing: Fixation Dehydration Clearing Infiltration Embedding

83. Fixation Most commonly used reagent for the fixation is 10% neutral buffered formalin The tissue bits should of the size of ~2x2cm & 2-3micrometer in thickness. These bits are then placed in metal cassettes or capsules which are then placed in the fixative. Tiny biopsies or small specimen can be wrapped in a filter paper and then put in a cassette & fixed. To help in visualization of small fragment of tissue during embedding, a few drops of 1% eosin is added

84. Dehydration Removal of free unbound water and aqueous fixatives from tissue components. Dehydrating agents are strong hydrophilic, possessing strong polor groups that interact with water by hydrogen binding. Dehydration should be accomplished slowly if concentration gradient is excessive, diffusion current across the cell membrane may cause cell distortion. The various dehydrating agents used are ; Ethyl alcohol Ethano acetone Isopropyl alcohol Methanol Glycol

85. Clearing agents act as an intermediary between dehydration & infiltration solution They should be totally miscible with both the dehydrating fluid & the embedding medium . It provide translucent appearance to the tissue Choice of a clearing agent depends upon the following : Rapid penetration of tissue Speedy removal of dehydrating agent Ease of removal by melted paraffin wax Minimal tissue damage Safety factors (low fammability , low toxicity) Cost and convenience Clearing agents: xylene , toluene, chloroform Clearing

86. Infiltration & embedding Most popular infiltration and embedding medium It is a mixture of long chained hydrocarbons produced by mineral oils It permeates the tissue in liquid form and solidify rapidly when cooled The paraffin wax increases the optical differentiation, hardens the tissue & helps in easy sectioning of the tissue. It is compatible to most routine, special stains as well as immunohistochemistry protocols Alternative embedding media: Resin, gelatin and celloidin Paraffin wax

87. Tissue Processing

88. Overnight processing schedule Station Reagent Time Temp 1 10% formalin 1 hour 38 degree C 2 10% formalin 1 hour 38 degree C 3 50% alcohol 1 hour 38 degree C 4 70% alcohol 1 hour 38 degree C 5 90% alcohol 1 hour 38 degree C 6 90% alcohol 45 min 38 degree C 7 100 % alcohol 1 hour 38 degree C 8 100 % alcohol 45 min 38 degree C 9 Xylene 1 hour 60 degree C 10 Xylene 30 min 60 degree C 11 Paraffin 1 hour 60 degree C 12 Paraffin 1 hour 60 degree C

89. Microwave processors Microwave irradiation fixes the tissue more effectively Large specimens which are soft to cut, can be irradiated for a brief period & then the tissue blocks are cut. The procedures of dehydration & impregnation is acceralated . Tissue antigens are well preserved following microwave irradiation. Microwave processing can be applied in electron microscopy & IHC.

90. Embedding Embedding involves the enclosing of properly processed, correctly oriented specimen in a support medium that provide external support during microtomy . It must fill the matrix within the tissue, supporting cellular components. It should provide elasticity and resisting distortion during sectioning

91. Embedding Centre Molten wax Cold plates Wax dispenser Wax flow adjuster Heated chambers Used lids Hot surface

92. Embedding process Dispense wax Align tissue Cool in place Cassette on Top-up wax Cool plate

93. Section Cutting ( Microtomy ) Microtomy is the means by which tissue is sectioned and attached the surface for microscopic examination The instrument by which this is done is called as a Microtome. TYPES OF MICROTOMES: Sliding Rotary Rocking Freezing Base sledge ultramicrotome Microtome knives: Disposable blades Glass and diamond knives

94. Paraffin section cutting Flotation water bath Slide drying oven or hot plate Forceps, brush and teasing needles Clean slide Slide rack Ice tray Diamond pencil Equipment required Section adhesives Albumin, gelatin , starch Poly-L-lysine 3-aminopropyltriethoxysilane (APES) Charged or plus slides (permanent)

95. Rotary Microtome It is the most commonly used. Block holder moves up and down while the knife remains fixed. Suitable for cutting of small tissues Parts of a Microtome ( Rotary ) : Block holder Knife clamp screws Knife clamps Block adjustment Thickness gauge Angle of tilt adjustment Operating handle.

96. Autostainer Staining (H & E)

97. Staining (H & E) Procedure Deparaffinize sections (2 changes of xylene , 10min each) Re-hydration (100%, 70%, 50% & 30%- 5min each) Wash briefly in distilled water. Stain in hematoxylin solution for 10 min. Wash in running tap water Differentiate in 1% acid alcohol for 30sec Wash running tap water for 1min. Bluing in 0.2% ammonia water for 30sec Counter-stain in eosin solution for 2 minute. Graded Dehydration (30%, 50%, 70% &100%- 5min each) Clearing with xylene (2 times- 5 min each) Mount with DPX

98. Mountants DPX ( Distrene Dibutyl phthalate Xylene ). Canada Balsam Colophonium resin Terpene resin