concepts of clinical testing Flashcards
patient management uses:
Medical examination
* Clinical history
– Presentingcomplaint – Pastmedicalhistory
* Physical examination
* Differential diagnosis
* Investigations
– Laboratoryand/orRadiology
* Diagnosis
* Treatment
– SurgeryorMedical
* Monitor outcome
– Diseaseandtreatment
labprtary investgiations uses:
- Clinical Biochemistry
- including endocrinology and
metabolism - Clinical Microbiology
- Haematology
- Immunology
- Medical Genetics
- the use of biochemical technique in the study of molular basis of disease and in diagnosis and management
- the aim is to provide an answer to a question posted about a patient
is ——-
clinical biochemistry
the process of the clinical biochemistry:
1- selection of appropriate —-
2- measuring of analytes in —-
3- interpetation of —-
- investigation
- body fluids
- results
the 3 phases includes:
1-pre analytical: —–
2- analytical : ——
3- post analytical : —
1-Appropriate investigation & sample collection
2-
* Method selection
* Quality control & uncertainty of measurement
3- interpretation of results
—- is the use of appropriate investigation and sample collection
-sample examples include:
- pre analytical
- Blood
Whole blood
Plasma
Serum - Urine
Spot urine sample
24-hour urine collection
- Blood
- Cerebrospinal fluid
- Sweat
- Faeces
- Sputum & saliva
- Tissue & cells
- Aspirates e.g. joint (synovial) fluid
1-pre analytical phase needs patient — such as:
2- the blood sample type such as:
1- patient preparation such as:
note of:
* Age of patient - Adult or child
* Time of day- Morning, afternoon or nighttime
* Fasting or post-prandial
* Exercise
* Medication
* Patient preparation
* Cleanse venepuncture site
2-:
- whole blood: serum clotted sample
- plasma : preservative , lithium heprin , EDTA
- transport of sample to lab: by porter + pneumatic tube transport
- laboratory info system (LIS): data entry
- sample preparation: labelling + centrifugation
analytical methods needs:
and it includes
- needs: method selection and faulty control & uncetentiy of measurements
in includes:
1- chromatography : GC or tandem -MS
2- immunological assays: enzyme linked immunosobenant assay ( ELISA)
3- spectrophotometric assay
4- nephelometric : light scatter
—- is used to provide unknown compounds by determining mass and matching to known spectra
it provides:
-mass spectrometry
it provides:
* elemental composition of sample
* masses of particles and molecules
* potential chemical structures of molecules by
analyzing the fragments
* Produces spectra of masses from molecules in sample and fragments of the molecules m/z
stages of mass spectrometry:
1- ioniser converts some of the sample into —
2- mass analyser separates ions according to their —-
3- detector records either — or —- when ions passes by or hits surface
- ions
- mass-to-charge ratio
- charge induced or current produced
—- is the application of science of enzymes to diagnosis of disease and monitoring of treatment and disease progression
- the measurement of enzyme activity or concentration in blood to:
- clinical enzymology
1- diagnosis: diagnose disease
2- complications: determine complications and extent of disease
3- monitoring: monitor disease progression
( check slide 16)
duchnenne musuclualr dystrophy:
- it results in a cycle of – and rapture of — in skeletal muscle
- this results in the release of the myocytes —-
- some of the myocytes cytoplasmic content including CK enzyme is released into —
- growth and rapture of myocytes
- cellular content
- blood
CK and the diagnosis of Duchenne muscular dystrophy:
- CK ( creatine kinase ) is an enzyme involved in —-
- presents in large amount in —- and —-found in — due routine turnover of skeletal muscle
- elevated – activity in DMD patient ( can be greater than 10x the reference range )
- elevated plasma CK activity in 75% of —- female carriers
- energy production
- skeletal and cardiac muscle
- blood
- plasma
- asymptomatic
other reasons of muscle damage includes:
Excessive physical exercise
- Marathon runners
- Recent strenuous exercise
Surgery
Skeletal muscle trauma Drug induced
1-in cellular damage - why do we measure enzymes ?
2- disadvantages:
- Relatively easy to measure compared with other proteins Can measure the enzyme product
- lack of tissue specificity and many enzymes present in more than one tissue
( check slide 23)
liver enzymes - plasma enzymes used to assess hepatic function includes :
- AST and ALT - aspartate and alanine aminotransferase
(formally called transaminases and still abbreviated to AST and ALT respectively) - ALP - alkaline phosphatase
- GGT - g-glutamyl transferase
liver diseases
1- pathological changes :
2- tests for liver disease:
1- pathological changes
- liver cell damage: acute hepatitis, chronic hepatitis, toxin action, alcohol abuse
- cholestasis: gallstones, cancer at the head of the pancreas Reduced mass of functioning cells (e.g cirrhosis, terminal hepatic failure) Infiltrative disorders - liver is invaded or replaced by nonhepatic substances such neoplasm or amyloid.
2- tests for liver diseases:
-Liver cell damage - enzymes - ALT, AST
-Synthetic function - prothrombin time, albumin
-Conjugating capacity - conjugated bilirubin
-Cholestasis - enzymes - Alk phos, γ-GT (also serum bilirubin)
hepatoceullar damage:
— is more liver specific than — however both enzymes are widely distributed in the body tissue but — is present only in small amounts in liver
— has cytoplasmic and mitochondrial iosoenzymes which tends to be released more than — in chronic hepatocellular disease
- ALT
- AST
- AST
- ALT
— is known as the failure of normal amounts of bile to reach the intestine
causes:
- cholestasis
- mechanical block (gallstone, tumour); liver disease; drug induced, e.g. phenobarbitol
more info : Alkaline phosphatase (ALP) and g-glutamyl transferase (g-GT) – normally “anchored” to membranes of hepatocytes, released in only small amounts in hepatocellular damage.
in cholestasis , the synthesis of these enzymes is induced and they are made –
soluble ( high concentration of bile acids )
- g-GT more liver-specific – ALP also raised in bone disease
-Changes in g-GT and ALP often parallel each other in cholestatic disease
alcoholic liver disease
- the abnormalities depend on – of the disease
- the biochemical abnormalities include:
- severity ( as hepatitis acute or chronic , cirrhosis )
- AST>ALT
-Raised γ-GT
-Increased mean cell volume (MCV) -Hyperbilirubinaemia
-Raised Alkaline Phosphatase
-Increased prothrombin time (INR) -Hypertriglyceridaemia
-Decreased K+, PO43-, Mg2+
—- are the parameters indicate that liver disease is due to excessive alcohol intake
Raised γ-GT
Increased mean cell volume (MCV)
-The appearance of a small amount of enzyme in the bloodstream from damaged tissue can be detected with —-
- enzymes must be — and conditions/buffer are important
great sensitivity , active
measurement of enzymes in blood - the factors affecting enzymes activity:
- Substrate concentration
- Temperature of reaction
- pH of buffer
- Presence of cofactors
- enzyme activators or inhibitors
- Urea & related compounds may disrupt hydrogen bonds causing inactivation
1—— are multiple forms of an enzyme
2- tissue specific ones is when certain genes may be expressed exclusively in — tissue as:
3- has cellular — within specific organelles as mitochondria and cytoplasm
-izoenzymes
- one
-as: LD , CK , alkaline phosphatase
- cellular localisation
izoenymes are multiple – of an enzyme that posses the ability to —–
they differ in —-
example:
- multiple forms
- catalyse enzymes characteristic reaction
- differ structurally - encoded by district gene
- example: creatine kinase and lactate dehydrogenase ( check slide 33)
— is known as the death of part of the whole heart due to a blockage of an artery
mycocardial infraction
cardiac biomarkers as —- are —- as they rise and then slowly fall over a period of time after an —
- CK-MB or troponin
- temporal
- infraction
measuring CK isotype levels:
- The different isotypes have different electrophoretic mobility.
- These can be quantified
- MM and MB forms predominantly
increased after infarct. - The brain isoform is not increased significantly
( check table 38)
plasma enzyme levels dependant on :
Rate of release from damaged cells Rate of damage to cells
Extent of cell damage
In the absence of cell damage, rate of release depends on:
- Rate of cell proliferation (malignancy)
- Degree of induction of enzyme synthesis
Rate of clearance (removal) from circulation
why measure enzyme activity in body fluids :
- A small increase in enzyme in the blood from damaged tissues can be detected with great sensitivity
- Monitor changes in substrate or product concentrations
- Easier than measuring protein directly as the signal is amplified from the reaction
- Can measure co-factor changes
- Enzymes are also antigens can be measured directly - bind to enzyme-specific antibodies
immunochemical technique is when – used to detect chemical substance of interest ( antigen )
- depends of – and high – of antibody for specific antigen coupled w ability pf antibody to – w antigen
- allows — and — of specific substance by variety of methods
- antibody
- sensitivity
- affinity
- cross link
- identification
- quantification
isotopic and non isotopic immunoassay:
Method of detection:
-Isotopic: Scintillation
-Non-isotopic: chemiluminescence, fluorescence, photometry
-Safety – hazards, handling & disposal
example:
1-Enzyme Immunoassays
Enzyme-linked Immunosorbent assays
- ELISA
2-Enzyme Multiplied Immunoassay Techniques - EMIT
( check till slide 49 pls)
enzyme multiplied immunoassay technique - EMIT:
1- homogenous assay to – drugs , hormones , and metabolites ( antigens )
2- patient sample added to a — containing —- and —
3- antibody – enzyme activity when no drug present
4- if there’s is a drug in the patient sample , this competes for the —
5- this — the enzyme site allowing the – to occur
- analzye
- tube
- labelled enzyme and antibody
- antibody
- opens
- reaction
In EMIT:
* No —— required- patient sample mixed with reagents
* The more drugs/hormone in the patient sample
* The more drug — to antibody (competing with bound Ab)
* The more enzyme is available for converting the substrate to the product
* A standard curve is used —- the amount in the sample
- seperation
- binds
- quantify
— is the interpretation of results and it includes :
- post analytical phase
- includes:
Validation of results
Review of internal and external quality assurance results
Internal – precision External - accuracy
interpretation of results :
1-Results are often reported as concentrations:
Concentrations change when:
-Amount of the analyte changes
-Volume of solution changes e.g. with dilution
(Beware differing units e.g. molarity vs. μg/ml)
2-Variability of results caused by:
-Analytical factors and Biological factors
3-Uncertainty of Measurement
- Estimates the difference between measured value and true value -
-describes a level of confidence in the measurement
4-Factors affecting uncertainty
- Sample collection and transport
- Calibration, precision and bias of assay
5-Reporting uncertainty of measurement
Plasma Sodium 141 mmol/L +/-0.64 mmol/L
( CHECK SLIDES FROM 52 PLSSS)
—– the range of values that represent 90 or 95% of reference population
reference range ( check slide 57)
Plasma alkaline phosphatase (total) activity Isoenzymes present in plasma derived from:-
Bone Liver Kidney Placenta
3rd trimester of pregnancy Intestine
following fatty meal ( check slide from 59)
- selection of enzymes for diagnosis or prognostic purposes depends on:
1-Distribution of enzyme among various tissues
-Tissue / plasma concentration gradient
-intracellular localisation
2-Convenience of enzyme assay
-Knowledge of plasma enzyme characteristics
-Half-life in blood
-Mode of clearance
why do we need to know all this - the lab results and reference ranges are affected by — such as:
- labarotary methodology as:
1- substrate and substrae concnetration
2- buffer and pH
3- temp
5- presence of enzyme activator/inhibitor ( check slide 63 -62)
