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Histology > Lecture 1 > Flashcards

Lecture 1 Flashcards

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1
Q

Histology

A

the study of tissue

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2
Q

Pathology:

A

the study of disease

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3
Q

Histopathology:

A

the study of diseased tissue

Overall, in the histopathology lab tissues are processed so that they can be viewed microscopically to detect malignancy or abnormality

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4
Q

Autopsy

A

Also called a postmortem examination or necropsy
 Used to determine the cause of death
Used to study diseased tissue
Performed by a pathologist or a pathologist assistant
 Autopsy specimens are sent to the histopathology lab for processing
 Tissue is similar to surgical specimens received and processed in the same manner as surgical specimens

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5
Q

Surgical Specimens

A 
oOrgans or parts of organ e.g. liver lobe, a kidney
oLymph node
oAmputated legs
oA breast
oProstate
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6
Q

Biopsies

A

A biopsy is an excision of a small tissue sample. Biopsies can be taken during endoscopy procedures, during surgery, or when a needle core biopsy is taken.
Biopsies are small pieces of tissue excision.
Needle biopsies
Uterine curetting or curettage
This includes tissue samples from cosmetic procedures.

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7
Q

Specimen Labelling

A 
Patient information
- Must have two unique identifiers such as:
Name and
 Date of birth
 Hospital I.D. Number
OHIP number
Specimen information
- Site/ type
- Specimen number
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8
Q

Requisition Labelling

A 
Patient Information
Name and
 Date of birth
 Hospital I.D. Number
OHIP number
Specimen information
- Site/ type
- Specimen number
Ordering doctor/physician
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9
Q

Accessioning of Specimens

A

All specimens must be sent with a requisition or an on-line order in the computer system
Verify that the information on the specimen container exactly matches the information on the paper requisition or on-line computer order entry.
Specimens are accessioned & given a surgical number
-This will be used to identify sample in the histology lab
-Usually starts with the letter “S” - surgical number
-The surgical number will be on the specimen label and on the patient report
-Many labs now use bar-coded labels

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10
Q

Specimen Rejection

A 
Discrepancies:
 Refer to the lab procedure manual.
 Will lead to rejection of the sample.
 Specimens cannot be discarded, however
 Must work with the source to workout labelling and clerical errors.
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11
Q

Fixed specimen

A
  • tissue arrives in fixative
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12
Q

Unfixed or “fresh” specimen-

A

Tissue arrives in no solution or in physiological saline

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13
Q

Safety precautions

A
  • Wear gloves and lab coats

- Treat all samples as if infectious

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14
Q

Fixation

A

Process where tissue placed into a chemical solution, undergoes physical and chemical changes which helps to prevent decomposition and distortion of the tissue
First essential step in histological processing of tissue.
Specimens that are yet placed in any fixative should be stored in a refrigerator (at ~ 4 degrees Celsius) to slow down decomposition.
For routine processing, tissue should be placed in fixative as soon as possible.
 Ideally the amount of fixative should be 10 to 20 times the volume of the specimen.
Most fixatives act by denaturing or precipitating proteins.

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15
Q

Specimen Issues

A

Specimen received with:

  • Inadequate amount of fixative
  • No fixative
  • Mislabelled
  • Labelling does not match the requisition
  • Specimen container leaking
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16
Q

Fixation - most important step

Purpose:

A

•To preserve the tissue in as lifelike a manner as possible.
•To stop autolysis and putrefaction.
-Most fixatives act by denaturing or precipitating proteins
- Tissues undergo hardening. This is beneficial as it allows the tissue to be handled and cut or sectioned more easily.
- Some fixatives help the staining of the tissue after it is processed. Some fixatives act as mordants .These help the dye used during staining to attach to the tissue being stained.
- Microorganisms will be fixed (and killed)

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17
Q

Fixation Autolysis

A
  • Decomposition due to the action of enzymes.
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18
Q

Fixation Putrefaction:

A
  • Decomposition due to the action of bacteria.
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19
Q

Formalin

A

Most common fixative used is 10% neutral buffered formalin

•Referred to as the universal fixative

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20
Q

Formalin/ Formaldehyde

A

Formaldehyde is a gas, soluble in water up to 37 to 40%by weight. This 37 to 40% w/v solution of formaldehyde is called 100% formalin.
It is an aldehyde with the chemical formula CH2O.
o It fixes tissue by cross-linking the proteins
o Formalin has the advantage of being a relatively cheap fixative

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21
Q

Fixation time for a specimen is affected by

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A

Size of the tissue
-Type of tissue

Time is needed for formalin to penetrate into the tissue and also for formaldehyde to bind to the tissue It cross links proteins in the tissue.

22
Q

B5 fixative

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A

B5 fixative is sometimes used for tissue such as lymph nodes and bone marrow biopsies because tissue fixed in this fixative shows good nuclear detail. This fixative contains formaldehyde and it also contains mercuric chloride.

B5 leaves mercury pigment in the tissue which must be removed during staining.

23
Q

Zinc Formalin fixative

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A
  • Shows good nuclear detail

- Now replacing the use of B5 fixative - fixative with mercury in it.

24
Q

Gluteraldehyde

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A

Fixative used in electron microscopy
It is a dialdehyde and fixes tissue by cross-linking the proteins. It penetrates tissue more slowly than formalin so tissue being fixed in glutaraldehyde should be cut into very small pieces.

25
Formalin– Safety Concerns
May cause sensitization by skin contact •Formalin fumes are very irritating to eyes and mucous membranes •Carcinogenic •Handle in a fume hood or in a well ventilated area and wear an approved respirator •Wear gloves, splash goggles
26
Grossing of Specimens
``` Gross description.  Done by a pathologist or a pathologist’s assistant or a technologist. dictated into a Dictaphone and transcribed later or dictated directly into the lab computer system. Includes : -Patient name. -Assigned surgical number. -Surgical procedure. Type of specimen -Size -Colour -Consistency -Any abnormalities seen ```
27
Grossing of Tissue | Large specimens:
Cut open or sliced. •Small representative sections no larger than 3-5mm square- is selected. • Placed in a tissue cassette for processing.
28
Grossing of Tissue | Biopsies or other small samples.
Biopsies are submitted “in toto” This means that the whole specimen is placed in the cassette for processing •Wrapped in lens paper or placed in between sponge and placed in cassette.
29
Cassettes
Grossed tissue placed inside cassette and lid closed Made out of plastic polymers resistant to histological solvents Has holes/ perforations to allow processing reagents to completely immerse tissue Surgical number imprinted on angled part of cassette. Cassette may also have bacode on it.
30
Cassettes | To label
``` Use pencil or solvent resistant marker •Can use cassette printer/ labeller •Many different colours of cassettes Can colour code: oFor type of fixative oPriority of specimens oBiopsy vs. Large specimen ```
31
Decalcification
The controlled removal of calcium from tissue. •Tissue containing calcium salts (e.g. Bone, scar tissue) is calcified and will be difficult to cut . •Tissue must be well fixed before it is decalcified otherwise the cell morphology will be destroyed by the decalcifying fluid.
32
Decalcifying Agents
These include: - Acids such as nitric acid , formic acid - EDTA which is a chelating agent ( binds to calcium ions).It is gentle on tissue but is very slow (takes 2-3 weeks) and is very expensive. - RDO, SURGIPATH - commercial preparations which are acid solutions + additives
33
Decalcification time
Gentle heat and agitation decrease decalcification time | •Over decalcification causes tissue distortion and poor staining
34
Tissue Processing steps
The next steps in tissue processing are : -Dehydration -Clearing -Paraffin wax impregnation or infiltration These are accomplished in an automatic tissue processor
35
Dehydration
The controlled removal of water from the tissue Dehydrating agents include alcohols such as ethanol ( most common) ,or acetone Tissue is placed in increasing concentrations of the dehydrating agent e.g.. 70% , then 85%, then 95% then 100% alcohol
36
Clearing
Removal of the dehydrating agent and replacing it with a solvent of paraffin wax Clearing agent o xylene -this is the most widely used clearing agent. It removes ethanol and makes tissue more transparent (i.e. “clears” the tissue). It is immiscible with water. -toluene -more expensive and more toxic than xylene. or Histoclear oMiscible with both the dehydrating agent with paraffin wax oClears the tissue – has a less opaque look
37
Infiltration
Removal of the clearant and infiltration with molten paraffin wax Melting points of the wax used varies from 54 to 58 degrees Celsius o During wax infiltration the temperature must be kept a few degrees (~ 5°) above the melting point of the wax. Paraplast – a mixture of paraffin wax and plastic polymers – is used in most histology labs
38
Automatic Tissue Processor
Can hold large number of cassettes 150 to 300) •Many different programs •Delay start time can be programmed •Heat and vacuum can be used to speed up processing Tissue cassettes are loaded in a rack which is placed in the retort Desired program is selected Overnight processing 12 to 14 hour program commonly used Stat biopsies can be processed in a 2 to 4 hour program
39
Maintenance and Quality Control of Tissue Processor
Regular change of reagents which must be recorded on a designated log General cleaning of processor surfaces Reagents used on the processor such as 70% alcohol have to be prepared o reagent change and rotation of reagents o hot water flush of formalin stations to remove any formalin precipitate o general cleaning of processor surfaces o recording maintenance performed in a designated log
40
Reagent/ Solution Preparation
``` Label all solutions according to WHIMIS regulations Labels should have: -Name of solution/reagent prepared -Date prepared -Precautionary labels and safety attire -Statement “ Refer to MSDS” ```
41
Processing Reagents-safety
Acetone, alcohol ( ethanol), toluene, xylene are all flammable liquids •Xylene and toluene are both toxic • Store in a sealed/ closed container •Keep away from heat/ flames or sparks •Use in areas with adequate ventilation or fume hood •Wear gloves, lab, coat, approved respirator, splash goggles
42
10% neutral buffered formalin
is a 3.7– 4% formaldehyde solution. It is used in histology as a routine fixative. Buffer salts are added to achieve a neutral pH. It is usually a phosphate buffered solution. It is sometimes referred to as NBF (neutral buffered formalin).
43
B5 safety concerns
o B5 fixative is toxic. It may be fatal if inhaled, absorbed through skin or swallowed. When handling staff should wear splash goggles, lab coat, gloves and a respirator or work in a fume hood or well ventilated area.
44
Cassette printer
This is an automated machine which is used to label tissue cassettes. The cassette printer may have a ribbon which needs to be changed and the daily maintenance needs to be carried out.
45
Use of different coloured cassettes
Tissue cassettes come in many different colours. Labs may colour code, that is, use a different colour cassettes to indicate: - Type of fixative used on specimen - Priority of specimens. All rush may be placed in cassettes of a certain colour so that they can be easily seen and handled more quickly. - Biopsy specimen vs. larger specimen
46
A good decalcifying solution must:
– Completely remove calcium salts – Minimize cell damage – Not affect the staining of the tissue – Work in a reasonable amount of time
47
Other fixatives
bouins fixatives | zenkers fixatives
48
Paraffin wax processing
refers to the method where the fixative is removed from the tissue and eventually replaced with paraffin wax. It involves the steps of dehydration, clearing and wax infiltration. During the embedding process the molten wax will harden to give support to the tissue during microtomy
49
Open System tissue processor
The specimens are placed in a basket which moves from one solution to the next.
50
o Closed System
The specimens are placed in a closed chamber (called a retort) and solutions are pumped into the retort, in sequence according to the program selected o Most tissue processors use gentle heat and vacuum and pressure to speed up the penetration of the reagents into the tissue. This reduces processing times o Some rapid tissue processors use microwave technology to reduce tissue processing time.
51
Clean or Purge Cycle:
This uses two reagents: first xylene then alcohol. The xylene removes the wax (which would harden and clog the lines). The alcohol removes the xylene so that the processor is ready to start a new processing program is commonly processed overnight in a 12 to 16 hour schedule

Histology (3 decks)

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