Histo Techniques

Question 1

what fluiddo samples from clinics, theatre and hospitals arrive in?

Answer 1

10% buffered formalin

Question 2

what is the minimum acceptance criteria for a specimen

Answer 2

Patient name, MRN, DOB, nature of the specimen or the specimen site, clinical information

Question 3

give some examples of large samples

Answer 3

colon, breast mastectomy, lung, kidney, ovary + uterus and prostate

Question 4

give examples of small specimens

Answer 4

biopsies
- core needle biopsies in breast, lung or lymph node
- stereotactic biopsies in the breast
skin biopsies
- punch, shaves, excisional, incisional, curettings, wide local excisions

Question 5

what are the 6 steps to the histology workflow

Answer 5

fixation, processing, embedding, microtomy, staining, diagnosis/result

Question 6

define fixation

Answer 6

attempts to preserve tissues in a life like manner and to prevent autolysis (self digestion) and putrefaction (microbial spoilage)

Question 7

what is the purpose of fixation

Answer 7

to prevent autolysis and bacterial decomposition, to coagulate tissues, to crosslink proteins, to facilitate downstream staining with dyes + other reagents (immunohistochemistry)

Question 8

what are the features of a good fixative

Answer 8

1. must kill the cell quickly without much distortion and keep the tissue close to living state as possible
2. penetrate cells rapidly and evenly
3. give good optical differentiation
4. inhibit putrefaction and autolysis
5. harden the tissue so that it is insensitive to other treatments
6. allow for multiple staining sessions

Question 9

what factors affect fixation

Answer 9

1.pH (6-8 is optimal),
2. temperature (most done at RT, but electron microscopy is done at 0-4 degrees)
3. the volume of tissue (larger tissues take longer to fix)
4. the duration of the fixation ( can take between 2-24hrs depending on sample/techniques)

Question 10

what is a simple fixative and give an example

Answer 10

where only one chemical is used- i.e aldehydes such as formaldehyde or glutraldehyde

Question 11

write a note on the function of aldehydes

Answer 11

create covalent bonds between proteins and tissues. protect secondary and tertiary structure of proteins + give strength and rigidity to tissue

Question 12

write a note on formaldehyde and gluteraldehyde

Answer 12

Formaldehyde: usually used in lab, typically mixed with other reagents to get a better fixative result for tissue samples
Gluteraldehyde: commonly used in electron microscopy- similar chemically to formaldehyde. used to fix small blocks of tissue for electron microscopy i.e renal biopsies. Inactivates all enzymes so it cant be used for downstream staining techniques

Question 13

write a note on oxidising agents

Answer 13

Osmium Tetroxide (OsO4)
- joins to side chains of proteins and allows cross link formation between proteins.
- stabilises tissue structure
- quite expensive and can be hazardous (eye nose and throat irritation)
- only substance that permanently fixes fat
- poor penetration power (suitable for small pieces only)
- main use is buffered fixative in electron microscopy

Question 14

write a note on denaturing agents

Answer 14

Acetic acid
1. counters shrinkage effect of other fixatives
2. preserves DNA well
Ethanol
1. rarely used on its own except in cytology
2. preserves proteins without denaturation
3. used in fixative mixtures to increase penetration of other fixatives

Question 15

write a note on mercuric chloride

Answer 15

1. in many compound fixatives
2. precipitates all proteins
3. rapid penetration and hardening
4. can leave a black precipitate in the tissue that has to be removed

Question 16

write a note on picric acid

Answer 16

1. binds to histones and basic proteins forming crystalline picrates with amino acids
2. precipitates protein
3. enhances cytoplasmic stains
4. explosive properties

Question 17

What isd 10% Buffered Formalin made of? What is a safety hazard of this?

Answer 17

10% formaldehyde 90% saline
Formaldehyde may cause dermatitid and its vapour causes irritation to eyes, nose and throat

Question 18

Examples of compound fixatives?

Answer 18

10% Buffered Formalin, Bouins fluid, Carnoys fixative

Question 19

What is Bouins fluid made of and what does it demonstrate?

Answer 19

Picric acid, formalin, glacial acid
Demonstrates glycogen

Question 20

What is Carnoys Fixative made of and what is its purpose?

Answer 20

Absolute alcohol, chloroform, glacial acetic acid
Preserves neurons and demonstrates nuclear detail well

Question 21

How long should histology large specimens, small specimens and electron microscope very small biopsies be fixed for?

Answer 21

Large - 7-24 hours
Small - 2-6 hours
EM - 3hours

Question 22

What is the gross report and what may the gross description include?

Answer 22

This is a report that contains info about the specimen- describes it
The gross description may include : size, shape, colour, weight, measurement of lesions/tumours, measurements of lesions to margins

Question 23

Processing tissues process

Answer 23

1. Dehydration using alcohols
2. Clearing using a clearing agent
3. Impregnation/ infiltration with a molten paraffin wax

Question 24

Benefits of using an automatic processor instead of doing it manually?

Answer 24

Decrease both time and labour and constant agitation during processing increases tissue chemical penetration

Question 25

What are the options for dehydrating the sample in tissue processing?

Answer 25

1. Alcohol method - consists of passing the tissue througha series of progressively more concentrated alcohol baths
2. Acetone method - used for most urgent biopsies - only small pieces of tissues can be used
   3.Dioxane method - rarely used as highly toxic vapour, high cost

Question 26

What must the clearing agent be? and what are examples of clearing agents?

Answer 26

Must be soluble with alcohol and wax
Clearing agents: Pro Par, xylene, benzene, chloroform, cedar wood oil