5&6 Analysis of Biological Specimens 1&2 Flashcards
1
Q
Describe the types of samples analyzed by the Core Laboratory.
A
- Whole blood (hematology)
- Plasma, Serum (Chemistry, coagulation)
- Urine (chemistry)
- CSF , body fluids (chemistry, hematology)
- Stool (chemistry)
2
Q
- Identify (3) common quantitative methodologies used to analyze samples.
A
-
Spectrophotometry and enzymatic rate reactions
- Measures changes in absorbance due to reaction with dye or coenzyme
- Eg Liver and muscle enzymes, glucose, some electrolytes
-
Electrochemical Sensors
- Measures electrical potential between two electrodes
- eg electrolytes, pH and blood gases
- Measures electrical potential between two electrodes
-
Immunoassay
- Uses antibodies with different labels to capture antigens in solution
- eg Cardiac markers and hormones
- Uses antibodies with different labels to capture antigens in solution
3
Q
Compare and contrast central laboratory-based models of sample analysis with newer models, such as point-of-care testing, including their advantages and limitations.
A
- Point of Care Testing (POCT) involves medical diagnostic testing performed outside the clinical laboratory in close proximity to where the patient is receiving care
- Fewer sample types can be analyzed
- Whole blood, urine, body fluid, stool
- Requires smaller sample volume (2uL blood (min 3ml), swab for fluids)
- Operators may not have formal training
- Small test menu (1-15 analytes)
- examples of POCT:
- Pregnancy tests
- Glucose tests
- Blood gases, electrolytes, kidney function, cardiac markers, coagulation etc
- Fewer sample types can be analyzed
- Lab-based models:
- Sample types:
- Whole blood, plasma, serum, urine, CSF, body fluids, stool
- Sample vol - greater than POCT (0.6mL min blood - 3-5mL preferred)
- Extensive test menu (10-100s analytes)
- Sample types:
4
Q
How is spectrophotometry and enzymatic rate reactions used in lab to analyze sample
Examples of analytes?
A
- Spectrophotometry and enzymatic rate reactions
- Measures changes in absorbance due to reaction with dye or coenzyme
- Eg Liver and muscle enzymes, glucose, some electrolytes
5
Q
How are electrochemical sensors used in the core lab?
examples of samples tested
A
- Electrochemical Sensors
- Measures electrical potential between two electrodes
- eg electrolytes, pH and blood gases
- Measures electrical potential between two electrodes
6
Q
How are immunoassays used in the core lab to analyze samples?
What types of analytes are typically analyzed?
A
- Immunoassay
- Uses antibodies with different labels to capture antigens in solution
- eg Cardiac markers and hormones
- Uses antibodies with different labels to capture antigens in solution
7
Q
Compare and contrast central laboratory-based models of sample analysis with newer models, such as point-of-care testing, including their advantages and limitations.
examples?
A
-
Lab-based models:
- Sample types:
- Whole blood, plasma, serum, urine, CSF, body fluids, stool
- Sample vol - greater than POCT (0.6mL min blood - 3-5mL preferred)
- Extensive test menu (10-100s analytes)
- Accurate and precise
- Often least expensive
- CON: Collection time
- Volumes
- Examples:
- Chemistry analyzer
- Hematology Analyzer
- Sample types:
-
Point of Care Testing (POCT) involves medical diagnostic testing performed outside the clinical laboratory in close proximity to where the patient is receiving care
- Fewer sample types can be analyzed
- Whole blood, urine, body fluid, stool
- Requires smaller sample volume (2uL blood (min 3ml), swab for fluids)
- Operators may not have formal training
- Small test menu (1-15 analytes)
- examples of POCT:
- Pregnancy tests
- Glucose tests
- Blood gases, electrolytes, kidney function, cardiac markers, coagulation etc
- Fewer sample types can be analyzed
8
Q
What are 4 advantages of POCT?
4 Disadvantages?
A
- ADVANTAGES:
- Collection-to-result turnaround time generally fast
- Blood sparing
- Decreases preanalytic errors
- Smaller space and power requirements
- DISADVANTAGES:
- Limited test menu
- usually the more expensive option
- Often results are less accurate
- (because non lab personal and assays have moderate-to-good performance)
- Harder to enforce compliance with regulations
9
Q
What are 5 advantages of Lab - based analysis?
3 Disadvantages?
A
- ADVANTAGES:
- Extensive test menu
- Automation = allows multiple tests and multiple samples run with minimal intervention
- Often the least expensive option
- Relatively easy to enforce compliance with regulations
- DISADVANTAGES:
- Collection - result turnaround time usually >30minutes
- Requires relatively large volumes of blood
- Analyzers require space, ventilation, a lot of power, water, liquid disposal
10
Q
A
-
Hematopathology (specimen types)
- peripheral blood,
- bone marrow,
- body fluids such as CSF
-
Histology (Anatomical pathology)
- Tissue of varying size from anywhere in body
- Foreign bodies (breast implants, synthetic grafts etc)
- Suspension of cells from body fluids (urine, effusions into cavities, fine-needle aspiration biopsies)
- cytology
-
Microbiology
- Specimen from just about any part of body
- bood
- tissue
- sputum
- stool
- urine
- CSF
- joint fluid
- body cavity fluid
- abscess fluid
- swabs …
- Specimen from just about any part of body
11
Q
List and briefly describe the steps in tissue preparation from grossing and fixation to microscopic examination.
Grossing
Fixation
Processing
Embedding, sectioning, Making slides
A
- Grossing
- The process by which specimen is examined macroscopically
- may occur before or after fixation
- includes identification (what, whose) measurements, weights, physical descriptions
- Selection of sections made into slides for microscopic analysis
- Sectioning and placing of tissue into blocks
- FIXATION
- Goals:
- Preservation of tissue
- Inactivation of infectious agents
- Hardening of tissue
- Required (for good fixation)
- Adequate amount (15-20X vol of tissue)
- Adequate time (6-8 hours)
- Thin enough tissue for adequate penetration (1mm/hr)
- Appropriate temperature (usually RT)
- Multiple fixatives available (eg formalin)
- Other fixatives available for specialized uses
- Choice depends on multiple factors
- Some tissues (eg bone) may need to be decalcified before sectioning, using an acid or chelator (eg EDTA)
- Goals:
- PROCESSING
- Done by big machines via preset programs
- Dehydration:
- water in tissue replaced by alcohol
- Clearing:
- alcohol is replaced by a substance that allows infiltation of the tissue by embedding medium (eg xylene - makes tissue transparent)
- Infiltration:
- Xylene replaced by embedding medium to make tissue stiff enough to be thinly sectionioned (paraffin)
- FFPE: Formalin-fixed, paraffin-embedded
-
EMBEDDING, SECTIONING and MAKING SLIDES:
- Infiltrated tissue removed from cassette, placed in a metal mold filled with liquid paraffin → block
- Orientation is important!
- Tissue block cut on microtome
- thickness varies
- Cut tissue floated on water bath and picked up on slide - stained and coverslipped
12
Q
List and briefly describe the steps in tissue preparation from grossing and fixation to microscopic examination.
Grossing
Fixation
Processing
Making slides
A
- Grossing
- The process by which specimen is examined macroscopically
- may occur before or after fixation
- includes identification (what, whose) measurements, weights, physical descriptions
- Selection of sections made into slides for microscopic analysis
- Sectioning and placing of tissue into blocks
- FIXATION
- Goals:
- Preservation of tissue
- Inactivation of infectious agents
- Hardening of tissue
- Required (for good fixation)
- Adequate amount (15-20X vol of tissue)
- Adequate time (6-8 hours)
- Thin enough tissue for adequate penetration (1mm/hr)
- Appropriate temperature (usually RT)
- Multiple fixatives available (eg formalin)
- Other fixatives available for specialized uses
- Choice depends on multiple factors
- Some tissues (eg bone) may need to be decalcified before sectioning, using an acid or chelator (eg EDTA)
- Goals:
-
PROCESSING
- Done by big machines via preset programs
- Dehydration:
- water in tissue replaced by alcohol
- Clearing:
- alcohol is replaced by a substance that allows infiltation of the tissue by embedding medium (eg xylene - makes tissue transparent)
- Infiltration:
- Xylene replaced by embedding medium to make tissue stiff enough to be thinly sectionioned (paraffin)
- FFPE: Formalin-fixed, paraffin-embedded
- EMBEDDING, SECTIONING and MAKING SLIDES:
- Infiltrated tissue removed from cassette, placed in a metal mold filled with liquid paraffin → block
- Orientation is important!
- Tissue block cut on microtome
- thickness varies
- Cut tissue floated on water bath and picked up on slide - stained and coverslipped
13
Q
List and briefly describe the steps in tissue preparation from grossing and fixation to microscopic examination.
Grossing
Fixation
Processing
Making slides
A
- Grossing
- The process by which specimen is examined macroscopically
- may occur before or after fixation
- includes identification (what, whose) measurements, weights, physical descriptions
- Selection of sections made into slides for microscopic analysis
- Sectioning and placing of tissue into blocks
-
FIXATION
- Goals:
- Preservation of tissue
- Inactivation of infectious agents
- Hardening of tissue
- Required (for good fixation)
- Adequate amount (15-20X vol of tissue)
- Adequate time (6-8 hours)
- Thin enough tissue for adequate penetration (1mm/hr)
- Appropriate temperature (usually RT)
- Multiple fixatives available (eg formalin)
- Other fixatives available for specialized uses
- Choice depends on multiple factors
- Some tissues (eg bone) may need to be decalcified before sectioning, using an acid or chelator (eg EDTA)
- Goals:
- PROCESSING
- Done by big machines via preset programs
- Dehydration:
- water in tissue replaced by alcohol
- Clearing:
- alcohol is replaced by a substance that allows infiltation of the tissue by embedding medium (eg xylene - makes tissue transparent)
- Infiltration:
- Xylene replaced by embedding medium to make tissue stiff enough to be thinly sectionioned (paraffin)
- FFPE: Formalin-fixed, paraffin-embedded
- EMBEDDING, SECTIONING and MAKING SLIDES:
- Infiltrated tissue removed from cassette, placed in a metal mold filled with liquid paraffin → block
- Orientation is important!
- Tissue block cut on microtome
- thickness varies
- Cut tissue floated on water bath and picked up on slide - stained and coverslipped
14
Q
List and briefly describe the steps in tissue preparation from grossing and fixation to microscopic examination.
Grossing
Fixation
Processing
Making slides
A
-
Grossing
- The process by which specimen is examined macroscopically
- may occur before or after fixation
- includes identification (what, whose) measurements, weights, physical descriptions
- Selection of sections made into slides for microscopic analysis
- Sectioning and placing of tissue into blocks
- FIXATION
- Goals:
- Preservation of tissue
- Inactivation of infectious agents
- Hardening of tissue
- Required (for good fixation)
- Adequate amount (15-20X vol of tissue)
- Adequate time (6-8 hours)
- Thin enough tissue for adequate penetration (1mm/hr)
- Appropriate temperature (usually RT)
- Multiple fixatives available (eg formalin)
- Other fixatives available for specialized uses
- Choice depends on multiple factors
- Some tissues (eg bone) may need to be decalcified before sectioning, using an acid or chelator (eg EDTA)
- Goals:
- PROCESSING
- Done by big machines via preset programs
- Dehydration:
- water in tissue replaced by alcohol
- Clearing:
- alcohol is replaced by a substance that allows infiltation of the tissue by embedding medium (eg xylene - makes tissue transparent)
- Infiltration:
- Xylene replaced by embedding medium to make tissue stiff enough to be thinly sectionioned (paraffin)
- FFPE: Formalin-fixed, paraffin-embedded
- EMBEDDING, SECTIONING and MAKING SLIDES:
- Infiltrated tissue removed from cassette, placed in a metal mold filled with liquid paraffin → block
- Orientation is important!
- Tissue block cut on microtome
- thickness varies
- Cut tissue floated on water bath and picked up on slide - stained and coverslipped
15
Q
List and briefly describe methods of morphologic evaluation of specimens (staining, microscopy, immunohistochemistry)
- Immunohistochemistry:
- Detect ______ by cells in FFPE tissue (______)
- Dif tissue types express different _____\_ =
- Use labelled _____\_ against these ______ to detect them in tissue
- Most often used to determine tissue of origin
- Cytokeratin → ______
- Vimentin → ______
- CD45 (LCA, leukocyte common antigen) → ______
- S-100 → ______
- Increasingly used to characterize cells for ______ and ______ purposes
A
- Immunohistochemistry:
- Detect protein expression by cells in FFPE tissue (formalin-fixed, paraffin-embedded)
- Dif tissue types express different antigens =
- Use labelled antibodies against these antigens to detect them in tissue
- Most often used to determine tissue of origin
- Cytokeratin → epithelium
- Vimentin → Connective Tissue
- CD45 (LCA, leukocyte common antigen) → Lymphoma
- S-100 → Melanoma
- Increasingly used to characterize cells for prognostic and predictive purposes
