histopath 1

histopathology

branch of pathology that involves the examination of tissues and cells at a microscopic level to diagnose diseases and understand their underlying structural changes

medical technologist

prepares tissues for microscopic examination

histopathologic techniques

deals with the preparation of tissues for microscopic examination

histotechnologist

histopathologic techniques are done by

role of histotechnologist

1. adhere to policies amd procedures

2. report problems immediately to the pathologist

3. perform proper histopathologic techniques

4. submit best quality slides to pathologisy with attention to proper numbering, labeling, sequence pf slides corresponding to sp request

5. assist the pathologist in maintaining quality practice in the histolab

• numbering

• fixation

• decalcification

• dehydration

• clearing

• impregnation

• embedding

• trimming

• section-cutting

• staining

• mounting

• labeling

coventional tissue processing

numbering

process of identifying the specimen without writing the patient’s name on the specimen tag

procedure, year, accession number

numbering contains:

fixation

first and most critical step in which specimen is placed in a fixative, prevents autolysis and stabilizes tissue to maintain cellular structures

10% neutral buffered formalin

most common fixative

37%

from what % of formalin is 10% neutral buffered formalin from

decalcification

optional depending on the specimen

nitric acid

most common acid in decalcification

• nitric acid

• hydrochloric acid

• formic acid

• picric acid

• acetic acid

• citric acid

examples of acids for decalcification

dehydration

removes the water and unbound fixative from the tissue

increasing

graded alchohols of — conventration are used in dehydration

ethanol

most common dehydrating agent

over processing artifacts

causes shrinkage, parched earth effect, and abnormal staining, dry brittle tissue

incomplete dehydration

impair penetration of cleaning agent leaving the specimen soft and non-receptive to laraffin wax infiltration

clearing

displaces dehydrating solutions making the tissue components receptive to the infiltrating medium

xylene

most commonly used cleaning agent

• xylene 20 mins

• xylene 20 mins

• xylene 45 mins

tissue is immersed in one to three different xylene immersions

impregnation or infiltration

permeates tissue with a support medium

paraffin wax

is liquid at 60 degrees and allowed to cool to 20 degrees (solid)

not more than 4mm thick:

to completely displace the clearing agents, a typical paraffin infiltration sequence of paraffin specimens — would be:

• pw 30 mins

• pw 30 mins

• pw 45 mins

incomplete infiltration

soft and crumby tissues

excessive

too much time in high temperature wax

embedding

orientation of the tissue sample in a support medium to create a tissue “block” siutable for sectioning

2-4 degrees above

melting point of the paraffin wax

correct orientation of tissue in the mold

is the most important step in embedding

down

tissues are embedded with the surface to be cut facing — in the mold

cold plate

in embedding, whole mold is placed on a — for molding

overheating

in embedding, hardening and distortion of tissues is caused by

infiltration

embedding uses the same chemical used in infiltration

trimming

happens before section-cutting

paraffin-embedded tissue

aims to completely expose the — in order to obtain a representative section

section-cutting

Embedded tissue are cut into sections that are thin enough to be placed on a slide usinb microtome

deparaffinization

removes paraffin wax from ffpr tissue sections

5-10 degrees celsius

ribbons are floated in warm bath at

60 degrees celsius; 15 minutes

Glass slides containing specimen are placed in warm oven at — for —

tearing, ripping, creases, holes for folding of sections

common artifacts in section cutting includes:

staining

uses chemicals of dyes that will bind or have affinity for certain components of the cells and extracellular components

hematoxylin and eosin

most commonly used stains

blue

hematoxylin dyes the nucleus

pink

eosin dyes the cytoplasm

mounting

Tissues are impregnated with transparent medium that has an index of refraction close to glass slide and tissue

canada balsam

most commonly used mounting medium

labeling

same as numbering

automated tissue processing

efficiently provesses large tissue volumes; replaces manual processing for better speed and consistency

1. water based tissue specimens

2. dehydrating agents

3. clearing agents

4. infriltrated with molten paraffin wax

provess flow of automated tissue processing

6hrs

Steps and timing vary based on tissue type and size; total time = —, often run overnight

carousel system

cassettes in a cage are agitated vertically through a series of glass beakers with different solutions.

single chamber

solutions are pumped in out ; more controlled and efficient

1. tissue density and thickness

2. agitation

3. temperature

4. vacuum and oressure

Factors that impact the duration of tissue processing and extent of infiltration

spongy tissues

density: — infiltrate faster like the lungs

hard or dense tissue

takes longer to be processed and extent of filtration

agitation

Increases flow of fresh reagents around tissues leading to faster exchange

vertical or rotary oscillation

mechanism of agitation

too high temperature

causes tissue shrinkage, hardening, brittleness (especially affects collagen)

too low temperature

reagents become more viscous leading to slower diffusion,

longer processing

2 - 3 degrees celsius

keep wax — above its melting point

high pressure

Helps force viscous media into dense/hard specimens

fine needle aspiration

Simplest, least invasive test and uses the smallest needle to remove cells from the area of abnormality

core needle biopsy

removes not only cells, but also a small of surrounding tissue, this provides additional information to assist in the examination of the lesion

incisional biopsy

lakes out even more surrounding tissue. It takes out

some of the abnormality, but not all

excisional biopsy

Removes the entire area in question

punch biopsy

considered the primary technique more for obtaining diagnostic full-thickness skin specimen

3 to 4 mm cylindrical core of the tissue sample

punch biopsy involves the use of a circular blade that is rotated down through the epidermis and dermis, and into subcutaneous fat, yielding

shave biopsy

small fragments of tissue are shaved from a surface

curettings

tissue is scooped /spooned to remove the tissue or growth from the body cavity such as endometrium or cervical canal

sing or dissociation

process wherein selected tissue specimen is immersed in a watch glass containing isotonic salt solution (nss), carefully dissected or separated and examined under the microscope

squash preparation or crushing

gprocess where small pieces of tissue not more than 1mm diameter placed in a microscopic slide and forcibly compressed with another slide or with a cover glass. vital dyes are placed at the slide and coverslip junction and absorbed through capillary action

smearing

techniques useful in cytological examination particularly for cancer diagnosis

streaking

use of an applicator stick or platinum loop applied in a direct

conservatively, whereas fatty specimens can be processed for longer than

or zigzag line

spreading

faterial is transferred in a clean slide and gently spread into a moderately thick film by teasing using an applicator stick recommended for preparations of fresh sputum, bronchial aspirates and thick mucoid secretions

pull-apart

two slides are pulled-apart with a single uninterrupted motion, useful in preparation of thick secretions such as serous fluid, conc. sputum, enzymatic lavage samples from git and blood smear

touch preparation

freshly cut tissue is brought into contact and pressed on

the surface of a clean glass slide

frozen section

normally utilized when a rapid diagnosis of the tissue in question is required

20 degrees celsius

tissue is frozen rapidly at — especially in intra-operative pathology to help the surgeon in choosing his next plan of action

fresh and unfiltered

for frozen section, the tissue should be sent to the laboratory —

-20 degrees celsius

cryostats should be preferably remain on all the time to maintain its teperature at

cold knife procedure

using a cold knife in a controlled cold environmenr

-40 to -60 degrees cesius

temprature of the knife in cold procedure

-5 to -10 degrees celsius

temperature of tissue in cold knife procedure

0 to -10 degrees celsius

temperature of environment for a cold knife procedure

cryostat procedure

refrigerated appratus used in fresh tissue microtomy consists of an insukated rotary microtome house in an electrically driven refrigerated chamber and maintained at temperatures near -20 C where microtome, knife, specimen and atmosphere are kept at the same temperature

-18 to -20 degrees celsius

Optimum working temperature of cryostat is

liquid nitrogen

generally, most widely used in histochemistry and during intraoperative procedures, and is the most rapid of the commonly available freezing agenta

carbon dioxide gas

used in cold knife procedure

isopentane

most excellent method for freezing muscle tissue

aerosol sprays

for small pieces of tissue except muscle