CELL PATH 1 Flashcards
time frame for when results are issued depend on?
distance needed to travel and workload
what happens in the gross room include workflow
specimens are analysed and reviewed -> specimen is received + logged into computer, gross description is made and dissection occurs if necessary- specimens then go to tissue processing
How do labs identify samples
samples are logged into the system, a bar code is given, the date and time received is labelled and if more than one sample is received from a patient numerical designation occurs[ A, B, C]
what is the minimal acceptance criteria for a sample
full name
other identifier [DOB or hospital number]
Details about the sample type is it a skin or bone
specimen request form must match specimen pot
comment on the importance of clinical details and give examples
accurate and complete clinical details provide a rounded picture of the case- clinical history also helps in diagnosis - i.e. if their was a history with a previous cancer and a liver biopsy is being examined metastatic cancer is a probable diagnosis and immunocytochemistry will be performed. or if there was a history with alcoholism - a liver cirrhosis is a likely diagnosis - special stains would be required
Dissection Area Characteristics
- Must be clean organized + ventilated
- Adjustable dissecting tables [inbuilt downdraught extraction - allows extraction of toxic formalin fumes
- Spillage kit
- Cutting board, ruler, scalper, forceps, handled knife and probes, blunt end scissors, balance, digital recording and photography, PPE [aprons], systems for cleaning
Preparation to dissect
- Review cases that need dissection
- Assign priority cases
- Assess case complexity
- Assess if specimen needs opening
- Adequate formalin levels
- Label cassettes [tissue type]
- Decide specimen type and categories
inking defines?
margins of resection # where tissue was cut out [indian ink]differential inking can also be done
Specimen Categories [comment on SOPS]
Specimens in A = transfer to tissue cassette =bone cores
B = transfer but need sampling, weighing or slicing =abscess
C - dissection + sampling [diagnostic assessment with preparation] [anus]
D - dissection + sampling [adrenal glands]
E- complex dissection + sampling [thymus]
- SOPS in place for dissection of particular specimen type + clinical history is taken into account
Definition of gross descriptions + what does it include
anatomic description which portrays macroscopic appearance of the specimen
1. Type + number biopsies received
2. Dimensions
3. Colour + consistency [tan yellow white]
4. Blood clots / foreign materials [soft hard / rubbery]
5. If Whole or part - assessed?
6. Tumour / abnormalities - location, number and gross description of tumour]
types of biopsy list them
Aspiration B -breast biopsy
Core biopsy - a type of percutaneous Biopsy - spring loaded gun = used
Cone Biopsy - diagnose cervical cancer
Endoscopic biopsy - Endoscope = used with sampling instruments
Punch Biopsy - skin rashes
LEEP
loop electrosurgical excision procedure
comment on paediatric specimens
- Take special care [difficulties diagnosing]
- Tumours from child usually small [need specialised procedures s- immunohistochemistry, flow cytometry, cytogenetics, electron microscopy and molecular genetics
give examples of gross-only examination samples
- Some samples = examined only grossly - non-tissue samples
- Bullets, implants and foreign bodies
- Tissues - teeth, ribs, fat vessels
- Foetal samples - miscarriages + still birth - strict guidelines
how long are histology samples stored post-final report
30 days
how long are fixed liquid cytology stored
21 days
definition of cellular pathology
alterations in cells + tissues caused by disease - can be infectious [i.e. helicobacter pylori can withstand stomachs acidic pH]
alterations in tissues can include
number, size + distribution of cells and molecular alterations [carbs, lipids, proteins + nucleic acids]
characteristic of cancer cells
MAPN
Metastasis - move from initial area of infection + replicate more
Apoptosis - cell death
Proliferate - increase in size
Neoplasia - excessive growth which leads to cancer
distinguish between histopathology + histology
histology - study of tissues [microscopic]
histopathology- study of changes in tissues caused by disease [microscopic]
what is clinical cytology// cytopathology
study of tissues within fluid
history of pathology - links by to
middle east during Islamic golden age
western Europe during Italian renaissance
What did the physician Avenzoar do
performed the first post-mortem
rudolf virchow is?
father of microscopy
what is formalin
A fixative + preservative
what are 3 sources of specimens
post-mortems, clinics, theatres
clinical cytology is involved in the preparation of what
fluid smears + fine needle aspirates
clinical cytology is used in what screening
cancer screening programmes
Tissue sample types - SLOBE
Scrappings - prostate
Lobe- lung
Organ - kidney
B- biopsies -kidney
E- excision- segment of the colon
Analysis of cells + tissue steps
- Prep for microscopy
- Histochemical methods are applied [#stains]
- Immunophenotyping of cells [ antibody detects antigen expression]
- Molecular analytical methods [changes in gene sequence #allows for early diagnosis]
- QC + microscopy + maintain chemical + sample materials
what happens if tissue is not preserved
autolysis + putrefaction can occur immediately
Types of tissue preservation
- Chemical preservation [most common] fixation in 1. formaldehyde 2. Glutaraldehyde or alcohol
- Freezing - snap freeze + store in freezer
- Heating - microwave preservation - Thermal [heating - protein coagulates which are abundant so tissue is held in place - boiling or microwave]
- Freezing - water crystalizes solid matrix is formed and tissue is hard - CO2 and liquid nitrogen
Chemical types of fixatives [most widely used] - done by coagulation + cross-linking - tissue samples are placed in chemical solutions which must permeate tissue [smaller specimens are permeated quicker + smaller]
Histochemistry
+ 4 e.g.s
study of chemical interactions in visualizing cell and tissue components 1. Dye stains
2. Metals [Gold + Silver staining]
3. Immunocytochemistry [antibody attaches label [binds to antigen] then allows visualization
4. DNA or RNA probe attaches label to nucleic acid - visualization
FIXATION definition
use of physical and chemical methods to prevent changes associated with tissue decay
Most tissues can be fixed but some can’t
- Muscle biopsies for enzyme histochemistry
- Skin + mucosal biopsies to investigate inflammatory skin conditions
[poor fixed tissues can compromise diagnosis [ will be visible once stained]
- Skin + mucosal biopsies to investigate inflammatory skin conditions
2 main reasons for fixation
prevents autolysis/putrefaction + maintains morphology + contents of tissue
what changes occur naturally to the nucleus
- KARYOLYSIS - nuclear fading chromosomes become denatured by DNAase and RNAase
- Pyknosis - DNA condenses - ends up as shrunken basophilic mass
- Karyorrhexis - pyknotic nuclei membrane ruptures and undergoes fragmentation
ALL of above lead to nucleus being dissolved and eventually leads to a dead cell with no nucleus
Ideal fixatives should do? give 6 examples
- Prevent autolysis + bacterial decomp
- Keep tissue in natural state + components
- Make tissue insoluble in liquids that’ll be used later in tissue processing
- Preserve tissue volume so it doesn’t swell
- Avoid excessive hardness
- Non-toxic + no-carcinogenic
give examples of cross-linking fixative + how does this cross-link form
Formaldehyde + glutaraldehyde - methylene bridge is formed between side and end groups of proteins
Mechanism of formalin
- Protein fixative
- Formed when formaldehyde [colourless gas] is mixed with H2O
- Is a liquid
- 10% formalin = 4% formaldehyde
- Commonly used in fixation
- Formaldehyde + H2O = made of methylene glycerol which is added to a protein and H2O is removed = METHYLENE BRIDGES = FORMED between 2 proteins bound due to m. glycerol and [protein reaction]
Benefits of formalin
- Cheap slow permeation
- Quick for small samples larger specimens= done overnight
disadvantages of formalin
- Biohazard
- Effects other biomolecule cells - Cytoplasmic streaming - carbs moved to side gives uneven staining [glycogen] - not suitable when testing glycogen storage disease
Control measure for Formalin// HEALTH + SAFETY ISSUES
- Toxic + carcinogenic
- PPE, containment level 2, well ventilation [formalin fumes are extracted when pot is opened]
fixation rate is divided into 2 parts
- Penetration rate [ time taken for chemical to reach innermost surface]
- Reaction rate [time taken for whole specimen to be fixated
FIXATION RATE - 0.75mm per hour = 8 hours for total specimen
- Reaction rate [time taken for whole specimen to be fixated
Glutaraldehyde
- CHO.(CH2)3.CHO
- Best morphological preservation
- Also cross-linking preservative
- Used in 2.5% ph Buffer
- Used for specimens undergoing electron microscopy [ not as effective permeation - so can’t be used on big specimens]
Comment on coagulant fixatives
- Alternative to cross-linking fixatives
- Dehydrate tissues [pull water out]
- Increased penetration rate
- Cause shrinkage [ poor preservation of mitochondria - not suitable for investigating disorders of mitochondria
E.G. ethanol, methanol, acetic acid and acetone
other fixatives names DPMO-C
- Osmium tetroxide - oxidising agent, secondary fixative - turns black [fixes lipid]
- Mercuric chloride - precipitates proteins - good staining quality - hardens tissue
- Picric acid - used in fixative solutions - changes charges of proteins side chain - good glycogen fixative
- Dichromate fixatives - secondary fixative - K dicromate = used to show chromaffin reaction [oxidation catecholamines synthesized by tumours - adrenal gland
Compound fixatives
Problems of fixation
- Under fixation
- Over-fixation
- Antigen masking [ foreign ag = no longer accessible for histochemistry
- fixated related deposits
List factors that affect fixation
- Buffer + ph [in low ph target groups on proteins = unreactive in formaldehyde] + the cross-linking effects of formaldehyde = reduced
- Duration of fixation + specimen size - time=distance fixative needs to penetrate through, compound fixatives components fixate @ diff rates. Gross specimens shouldn’t be allowed to sit in fixative rather suspended
- Temperature [ molecules diffuse quicker at an increased temp i.e formaldehyde penetration increases @ higher temp - tissue processors = enclosed @ higher temps 60-65 [but its controlled due to danger of fumes]
- Concentration of fixative -need to get it right as it varies from fixative -> fixative if formalin conc is greater than 10% = 10% hardening + shrinkage [ineffective fixation] + ethanol concen less than 70% will fail to remove water
- Osmolarity of fixative [best results when slightly hypertonic [ higher solute concentration] - if hypot-tissue will shrink -too hyper- tissue will swell
DECALCIFICATION
calcium must be removed from bone samples after fixation
what is decalcification
the removal of calcium from bone samples after fixation
Tissues is fixed [list following steps in x4
- Tissue processing
- Microtomy [cutting + paraffin wax]
- Staining
- Microscopy
what is tissue processing + give overview
removal of H2O- which is replaced with a medium - tissue is solidified and can be cut by microtome [done by tissue processor
1. Dehydration - H2O + fixative = removed
2. Clearing - dehydrating fluids = removed - tissue can now receive infiltration medium
3. Infiltrating - tissue is permeated with a support medium [paraffin wax]
4. Embedding - tissue = orientated in medium -> solidifies
aims of tissue processing
- Medium used = stable, firm, non-hazardous [i.e. wax]
- Helps ability to cut thin section
- Allows good preservation of tissue morphology + their contents
- Long term preservation
