Tissue processing & sectioning

Biopsy: examination of tissue taken from living body (gross &microscopical examination).

Autopsy: examination of dead body

Disease: any abnormality in the structure or function of an organ or tissue .

Types of biopsy

1) Incisional biopsy: a portion of tissue from a large lesion is taken-only diagnostic

2) Excisional biopsy: the entire lesion is removed with a margin of adjacent normal tissue-diagnostic & therapeutic.

3) Punch biopsy: by biopsy forceps in the uterus ,cervix, oral cavity, esophagus.

4 (Core needle biopsy: by wide bore needle used percutaneously for sampling of internal organs.

5 (Curettage biopsy : for diagnosis of uterine diseases .

Multiple excised masses ( excisinal biopsy)

Large bulky well defined mass removed totally ( excisinal biopsy)

Segment of colon removed with central mass ( excisinal biopsy)

Kidney : (excisinal biopsy)

Uterus with enlarge ovaries ( excisinal biopsy)

Handling of biopsy:

Once a biopsy is taken, it should be put in plastic or metal container with adequate amount of fixative ( 10% formalin) which causes rapid denaturation of cellular proteins & prevents autolysis. It should be sent to the lab with a request form including patient’ s name, age, sex, short clinical notes, type of biopsy, name of tissue submitted & findings of operation.

General principles for gross examination:1) Proper identification & orientation of the

specimen.2) Place the specimen on a cutting board &

record all the following data• Type of specimen• Dimension ( in centimeters)• Weight• Shape• Consistency• Surgical margins whether included or not

involved by the tumor.

Histopathological techniquesDeals with tissue deals with preparation of

tissue for histopathological examination, the aim of these technique is to preserve microscopic anatomy of tissues & to cut tissue in very thin sections( 4-5 microns) this is achieved by passing tissue in a series of process

Tissue processing can be done manually or mechanically & includes the following processes

1) Fixation2) Dehydration3) Cleaning4) Embedding5) Cutting6) Staining

Tissue processor used for biopsy processing through passing the biopsy into multiple steps

FixationMost fixatives act by denaturating or

precipitating cellular proteins which form meshwork that hold other structures & prevent autolysis.

The most widely used fixative is 10% formalin.

DehydrationIs removal of water molecules from tissues

and is achieved by graded alcohol.CleaningAlcohol replace water in the tissues, removal

of alcohol from tissues is by Xylene which creates empty tissue spaces to be infiltrated by wax.

Embedding with waxParaffin wax is used for embedding of tissue

which form tissue blocks after cooling the it can be trimmed into thin sections (4-5microns) using the microtom the sections are placed on glass slides and become ready for staining

Embedding with wax

Tissue blocks are trimmed into thin sections (4-5microns) using the microtom

sections are placed in water bath for opening of folded tissue

Sections are placed on glass slides and become ready for staining

Hematoxylin & eosin is the most widely used stain in histopathology

Nuclei appear dark blueCollage & cytoplasm appear pinkKeratin appears pink to red

Special stains1) PAS ( periodic acid schiff) stain

glycogen & mucin 2) Congo-red for amyloid3) Sudan-black for fat4) Giamsa stain for helicobacter pylori

H-pylori stained dark blue with giamsa stain

Amyloid deposits stain orange-red with Congo Red stain

Frozen sectionIn this technique the tissue is frozen rapidly

(using cryostat) to -20ºC ten sections are cut and stained (without passing in the steps of tissue processing) so that tissue can be examined microscopically within 5-10 minutes of removal from the body

It allows rapid diagnosis of the nature of the

lesion whether benign or malignant to decide the next step in surgery.

All laboratory staff should be informed and all preparations should be completed before arrival of tissue

Cytology

Is the study of cell ( normal or diseased altered cell) obtained from various sites of the body. It allows rapid diagnosis often within minutes.

Cells examined by this process are collected by one of the following methods:

1) Exfoliated cell2) Cells removed by brushing or scraping3)Removal of cells from deep tissue ( by aspiration)

Exfoliative cytology Spontaneous shedding of cells derived from

lining of cavities where they can be removed by non invasive methods e.g. vaginal smear, sputum examination, voided urine, body fluids, nipple discharge

Abrasive cytologyCells specimens are obtained from

superficial scraping of the lesion e.g.cervical scraping (pap smear) buccal mucosal smear skin scraping of various lesions

Fine needle aspiration cytology

Cells are aspirated from deep non surfaces organs or masses e.g.

Beast massThyroid nodulesPalpable lymph nodesInternal organs like liner and kidney

Technique of cytology1) using a needle and syringe to obtain cells

from a mass. exfoliatied material is sprayed on a slide directly

The needle is gently introduced through the skin into the mass ( while the mass is fixed in between fingers and thumb) and is moved in different directions while applying negative pressure onto the syringe with continuous suction of material through out the process, then the needle should be withdrawn gently from the mass

2) Smearing collected material on a glass slide3) Immediate immersion of the slide in a fixative

( 95% ethanol) to avoid dryness of material 4) Applying a stain5) Examine under the microscope

Palpable lymph node in the neck can be examined using fine needle aspiration cytology

Aspiration of material from cervical mass while applying negative pressure

Spraying aspirated material on a glass slide

Immediate fixation of material in ethanol

Staining of slides

Cytology is the study of cell

Indications of cytopathology

1) Diagnosis of malignancy2) Diagnosis of precancerous changes e.g. cervical

atypia.3) Detection of inflammation and pathogenic agents

e.g. fungal or parasitic infection of vagina4) Study of hormonal patterns and gonadal function

e.g. examination of squamous cells in vaginal smear which are under influence of onerian hormones to assess overian function in infertility.

5) Identification of sex chromosome in newborn with ambiguous genitalia, buccal smear is used as a source of cells.

Limitations of cytology

1) The nature of lesion is not so obvious as in a histological section.

2) Difficulty in identification of the exact site of lesion e.g. malignant squamous cells shaded in sputum may arise from buccal mucosa, pharynx, larynx and bronchi.

3) The size of lesion cannot be approximated by cytology.