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What can you use diagnostic testing for?
- evaluate health
- diagnose disease
- decide on appropriate treatment
- monitor effects of treatment
Define sensitivity and specficity
Sensitivity: fraction of patients who have disease that is correctly predicted by the test
Specificity: fraction of pts who do not have disease that is correctly predicted by test
What are the axes on Reciever Operating Characteristic curves?
Sensitivity (y) vs 100-specificity
Define PPV and NPV
What do these values depend on?
PPV: likelihood of a correct positive result
NPV: likelihood of a correct negative result
These values depend on disease prevalence
Impact of an imperfect test
- 5000 per 100,000 received biopsies
- 1600 per 100,000 treated for prostate cancer
- only 60 deaths reduced
Good example of sensitive/specific test
PET imaging of AD.
18F-florbetaben binds B-amyloid; can image the brain
- PET more sensitive than genetic screening
- 80% sensitive and 90% specific
Define reference value and interval
Define percentile and interpercentile
reference should be people who resemble the patient in all respects, except for the characteristics being tested
- percentile = 99% of reference values lower than that value
- interpercentile= bounded by percentile values
Diagnosis of AMI
changes in cardiac biomarkers (troponin) abouve the 99th percentile of URL and evidence of ischemia (ex. ECG, pathological Qwaves, )
Principles of Immunoassay(ELISA)
monoclonal antibodies specific for the analyte are used to bind the analyte in a sample
TSH assays are usually what type?
- primary antibody recognizes a-subunit
- secondary antibody recognizes b-subunit
always calibrated using a WHO TSH reference preparation.
results in milli-IU/L of serum based on a comparison to a bioassay for TSH
Describe the Biocheck TSH enzyme assay
1) Immobilized anti-TSH mAB incubated with TSH
- 2) Wash
- 3) Add goat anti_TSG-hoseradishperoxidase (labelled)
- 4) ADD TMB
- 5) HRP catalyzes TMB-> Coloured-TMB [absorbs at 450nm]
What is a common problem with sandwich-type assays, and how does this relate to TSH assays?
- Non-linearity due to insufficient primary anti-TSH antibody coated
- onto microELISA plate
- common problem with 'sandwich' type assays
- I.e. at high concentrations, not all TSH will bind so the curve
Explain the T4 immunoassay
- competes between T4* and T4.
- T4*-AB1 complex is separated from free T4* and measured
- signal obtained is plotted vs concentration of unlabelled T4 for a series of
- known concentrations of T4
- unknown T4 in plasma determined by comparison with standard.
Explain Autobio Chemilluminescence Immunosassay for T4
- 1) Immobilized anti-T4 mAB incubated with fixed amount of T4*
- (with HRP) and sample of T4
- 2) Incubate at 37 degrees for hour with chemilluminescent
- 3) Wash
- 4) Measure relative light units (conversion of substrate to
What does albumin tell you about liver disease?What does ALP tell you about liver disease?AST/ALT
- albumin tells you about cute/chronicity of disease
- AST/ALT tells you whether the damage is hepatocellular
- ALP tells you whether the disease is cholestatic
What does AST do?What does ALT do?which one is more specific for liver?
- transfers aminogroup from aspartate to make glutamate
- ALT: transfers amingroup from alanine to make glutamate
- ALT is more specific to liver (also in heart/muscle/kidney), rarely elevated
- except in hepatocellular disease
- AST is widely distributed in body (mostly heart, then liver, then muscle, kidney)
explain the assay for AST and ALT
- of coupled enzyme assay
- Assay rxn + Indicator rxn
- indicator rxn results in conformation change in NADH-> NAD+ changing
- absorbance signal
- Indicator rxn enzyme must be in excess, so only limiting factor is the amount
- of enzyme of interest
Explain how NADH-> NAD conversion can be used to measure AST/ALT
Rate of change of absorbance at 340 nm/minute is proportional to the amount of NAD+ which in turn is proportional to the amount of substrate converted/minute
NAD+ + H+ + 2e- -> NADH
- as long as substrate is in excess the velocity of the rxn is 0 order, and is
- proportional to amount of enzyme present.
- enzyme+ substrate -> enzyme + product
- (should know the michaeles menten eqn too)
Explain what alkaline phosphatase is (ALP)
- monoester + H2O -> alcohol + phosphate
- several isoforms, including bone, intestines, liver, kidney
- most ALP in serum originates from liver and bone
- increased serum ALP associated with liver dysfunction or bone osteoblastic
- increases synthesis of ALP in response to cholestatic disease!
Explain the ALP assay
- assay that relies on coversion of 4-nitrophenylphosphate (colourless) to
- 4-nitrophenoxide by ALP which is measured at 405nm (yellow)
(should know the rxn)
Serum albumin assay
- colourimetric assay that relies on the binding of 'albumin-specific' dyes to serum albumin.
- measure absorbance at 610-620 nm
bromcresol green or bromcresol purple (more specific for albumin)
remember that albumin tests for acute/chronicity
When do peak transaminase levels peak?What type of medication interfere with transaminase assays?How should the serum/plasma be stored?
- peak 7-12 days after hepatic injury, then decrease within 3-5 weeks (unless
anti-coagulants can inhibit the assay
serum or plasma samples should be stored at 4degrees and not hemlyzed
What is proteinuria?How is proteinuria measured?
passage of serum proteins (especially albumin) into urine by glomeruli.
- measured by turbidimetric method follwing precipitation with benzethonium
- chloride or by dye-binding
- i.e. urine sample protein is preciptated then turbidity measured
Where is creatine made? Describe the creatine assay
- creatine synthesized by kidneys, liver and peancreas, then transported to muscle and
- brain where it's phosphorylated.
Explain the assay for SrCR
- Jaffe RXN
- Picric acid+ Creatinine ->(in OH-)
- Picric-creatinine complex (red-orange colour)
- absorbed at 490-500 nm
some interference by compounds can increase apparent SrCr (ex. ketones)
Alternative rxn for SrCr
couples enzyme rxn involving creatininase that generates H2O2 which converts a substrate into a coloured product that can be measured.
What are some technical problems with the Jaffe Rxn?
- not specific for Cr
- small intereferences from protein/glucose/ascorbic acid
- some assays compensate by substracting a fixed from normal value
- some assays will overestimate by 20%
enzyme based assays are more specific but still some interference
What are some differences between MDRD and C-G?
MIDRD more accurate than cockroft gault and doesnt require patient weight, but both use SrCr parameter
What is therapeutic index? For which drug my peak and trough concentrations be more reflective of efficacy and toxicity?
difference between concentration that is effective and concentration that is toxic
What are some criteria for TDM? what are some examples of TDM drugs?
- narrow therapeutic index
- correlation between plasma/serum concentration and efficacy/toxicity
- variability in inter-individual pk (liver/kidney disease or non-linear pk)
- absence of biomarker for therapeutic response
- potential DI that could affect plasma/serum
Digoxin, gentamycin, vancomycin, valproic acid, lithium, theophylline, tacrolimus, lidocaine, phenytoin, cyclosporine
What are some advantages of immunoassays for TDM?
What is the difference between a heterogenous and a homogenous immunoassay?
- gold standard, most comon
- easily automated
- relatively specific for drug
- heterogenous require seperation of AbAg* complex for measures
- homogenous do not require seperation
3 major advances made by Dr. Yalow in regards to peptide hormones?
- 1. allows measurement of really small levels of hormone (picomolar range)
- 2. technique was specific for the binding of specific molecules (as the molecule was in competition with itself in a radiolabelled format)
- 3. Found that non-human insulin was immunogenic; thus even small peptides can be immunogenic.
Principles of Radioimmunoassay (RIA).
how is digoxin affected by this?
- 1) antibody-bound radiolabelled drug on assay surface incubated with serum
- 2) competition between antibody-drug and radiolabelled-drug-antibody complex
- 3) measure antibody-bound radiolabelled drug
ex. of heterogenous assay
digoxin-like immunoractive factor is present in pregnant women and neonates, as well as other substrates with steroid structure (cortisol, estradiol) can interfere
"apparent-digoxin" shows on assay, even though women not on digoxin
What are the principles of the EMIT assay? [enzyme-multiplied immunoassay]. what is an example?
- 1) antibody bound to drug-enzyme conjugate on assay surface, incubated with serum/plasma (w/ drug of interest)
- 2) Drug will displace the drug-enzyme conjugate
- 3) free drug-enzyme converts the substrate which produces a colour
- 4) measure colour
Is a homogenous assay: no need to seperate as you can just measure colour
Explain the gentamicin assay. what type of assay is it?
- gentamicin conjugated with G6PD
- glucose as enzyme substrate
- conversion of glucose to gluconolactone by G6PD results in production of NADPH which is measured at 340 nm.
What is a chemiluminescence assay? Give an example
- emits fluorescence for long periods of time
- principle: acridinium esters undergo a chemical rxn when exposed to H2O2 which produces light.
ex. phenytoin CIA
- 1) Phenytoin-acridinium conjugate bound to antibody on microparticle incubated with phenytoin from serum/plasma
- 2) measure proportion of these forms indirectly by measuring chemiluminescence for phenytoin-acridinium conjugate bound to microparticle
What are primary patient considerations?
What are secondary patient considerations?
- Patient considerations:
- Primary survey of patient:
- patient history, initial physical examination, ancilliary tests and general lab tets
- Secondary survey of patient condition:
- more detailed Px, examination of items brought in with patient, more specific lab tests
What are the principles of POC drug-screens? what are somelimitations of POC drug screens?
- in urine competes for binding with fluorescently labelled drug to channel in
- cartridge. fluorescence measured in POC meter
- drug concentration must exceed a certain threshold value
- quantification requires other assays
- Limitation: drug may be present in concetrations lower than threshold value;
- does not indicate toxicity of drug, just presence; no quantitiative measurement
Explain the enzymatic ethanol assaylevels of ethanol intoxication
Ethanol + NAD+ -> (ADH) Acetaldehyde+ NADH
Rate of change of NADH is measured at 340 nm
euphoria/excitement; confusion; stupor; coma; death
Explain the osmolar gap ethanol assay
- freezing point depression of plasma sample in an osmometer
serum osmolality increases by 10 mOSm/kg for each 0.06g/100mL of ethanol
osmolar gap: measured osmolality- calculated osmolality
How migh tyou measure other alcohols in the serum/plasma?
Gaschromotagraphy with flame ionization detector
Explain the acetaminophen assay.How is acetaminophen toxicity assessed?
Acetaminophen-> p-aminophenol -> blue colour
- Rumack-Maathew nomogram assesses clinical significance of acetaminophen
Explain the Rumack-matthew nomogram
hepatic toxicity of acetaminophen due to metabolite NAPQI. Probability of hepatictoxicity depends on time since ingestion andplasma concentration ofacetaminophen defined by nomogramAceta. conc (y) vs. Hours after ingestion (x)note that there is a 4hour delay between ingestion and assessment of clinicalsignificance.
explain the Salicylate assay and the alternative method
Salicylate + Fe3+ -> violet complex (absorbed at 540nm)
called the Trinder rxn
- salicylate + NADH + H+ -> (salicylate hydroxylase) catechol +
measured NADH ROC at 340 nm
Automated chemistry analyzers can be used for
drugs of abuse and drugs requiring TDM
What is a biomarker?What are some roles for biomarkers?
Characteristic that is objectively measured and evaluated as an
- indicator of normal biological processes, pathogenic processes, or
- pharmacologic responses to a therapeutic intervention
- sceen for early detection
- aiding in diagnosis of cancer
- predicting survival from cancer
- predicting therapeutic responses
- tumour staging (stages 1-4)
- Detecting recurrence
- monitoring effectiveness of treatment
Carcinoembryonic assay (CEA)
- cell surface glycoprotein adhesion molecule
- elevated in serum in colorectal, lung, gastric, breast
- some false positives (liver disease, bowel disease, smokers!) as
- well as false negatives. Also found in embryonic tissue
- Serum CEA decreases w/ successful treatment of colorectal cancer
- and rises with recurrence.
Why is CEA not a good screening tool? What is it good for?
0.6%of smokers have elevated CEA compared to normal, but it is good to measureresponse to treatment if you know they have the cancer
CEA for detection of recurrent CRC
- 314 pts surgically treated for CRC and monitored over 3 years with CEA levels
- CEA measured by immunoassay
- At cut off level:
- sensitivity= 50%
- specificity = 99%
- PPV= 74%
- NPV= 92%
thus u can be sure that if test was negative,there was no recurrence of CRC
- found elevated in prostate gland but also some false-postives (BPH) as well as
- false negatives.
requires follow-up with biopsy for diagnosis
- sensitivity = 78%
- specificity = 33%
glycoprotein expressed mainly by ovarian cancer, but also, endometrial, pancreatic, lung, breast, colorectal.
- increased in serum as disease progresses from stage 1 (50% of pts) to stage 2-4
- at >35 u/L, sensitivity = 95% and specificity= 82%
- detection of recurrent or residual disease after treatment but not for
- screening (except in high risk women)
- glycolipid secreted by normal pancreas and biliary duct cells
- elevated in pancreated cancer, gastric, hepatobilliary and other cancers, but
- also pancreatitits
- sensitivity= 69-93%
- specificity = 76-99%
often patients dont seek treatment until cancer is advancedd
HER2 how are people selected for treatment?
- example of personalized cancer therapy biomarker
- over-expressed in 20-25% of breast cancers and is the target for
- tastuzumab (herceptin)
- patients selected for hercepted treatment based on
- immunhistochemical (IHC) sstaining of a tumour biopsy for HER2 or by
- fluoresence in situ hybridization (FISH)
Immunohistochemistry for HER2
what is a postiive cutoff for HER 2?
- 1)Add primary antibody (recognizes HER2)
- 2) Wsh
- 3) Add secondary antibody conjugated to an enzyme (HRP)4) add DAB substrate and H2O2, forms a colour by enzyme rxn
- Positive staining on greater than 30% of tumour cells (which is a 3+ rating)
FISH for HER2
- in situ hybridization
- denature chromosome on which HER 2 is located
- spliti double strands and hybridize with DNA from a fluorescent probe
- image the fluoresence
- -pink dots= her 2 gene; control gene= green
- ratio of her-2 to control gene should be 1
- if ratio exceeds 2.2, the patient is eligible for herceptin
Imaging as a biomarker for HER2
- give 115-IN labelled pertuzumab
- see if drug accumulates, if it does > patient can be treated
- imaging using spect/CT
Estrogen and progesterone receptors for breast cancers
- ER and PGR are measured in breast cancer by IHC staining of a tumour biopsy.
- Positive stains of >1% of tumour cell nuclei is considered ER positive but
- response to anti-estrogen therapy is most common with > 20% positive
- Can use tamoxifen or aromatase inhibitors (letrozole) to treat ER+ breast
- mammaprint microarray analysis probes 70 genes to seperate early stage 'node-negative'
- breast cancer patients into low or high risk for recurrence. pts at hihg-risk
- may need to be treated with chemotherapy.
essentially mammaprint stratifies pts who will benefit from chemotherapy.
- take tumour, isolate mRNA for a host of genes (70 genes) associated with
- tumour. Isolated mRNA is fluorescently labelled and hybridized with a
- complimentary piece of DNA, if genes are over-expressed-> more fluoresence.
- probes 21 genes to derive a recurrence score for ER-Positive
- oearly stage breast wcancer which aids in decision-making about
- treatment. (chemo, hormonal, both types)
- performed by reverse transcriptase-PCR
CD20 in non-hodkin's lymphoma
- phosphoprotein expresed on normal b-cells and on non-hodkgin's b-cell lymphoma.
- target for mAB rituximab
How does flow cytometry for CD20 work?
- Flow cytometry analysis of CD20 expression on B-cells
- based on bidning of fluorescently-labelled antibodies to cells and
- their sorting by fluoresence intensity
- more than 90% of B-cell lymphomas express CD20, thus are sorted
- differently than normal cells.
BCR-ABI translocation in CML
- 9-ABI gene; chromosome 22- Bcr gene
- in chronic myelogenous leukemia, there is a translocation of the AbI gene from
- chromosome 9 to chromosome 22 in 90% of cases, which creates BCr-Abi fusion
- gene product is a constitutively active tyrosine kinase for proliferation of
- treat with imatinim
tyrosine kinase inhibitor which competes with ATP for
- phosphorylation sites on BCR-ABL protein in CML. Also used fo GI stromal
- tumours (phosphorylation activates the receptor)
- imatinib has much higher survival rates than chemo
How can you measure BCr-AbI protein in CML?
- uses a threshold value.
- take mRNA, convert to cDNA using reverse transcriptase
- use a TaqMan probe (5'end is radiolabelled, 3' end has a quencher)
- Taqman polymerase knocks off nucleotides and when the radiolabel is knocked off
- it will fluoresce as it sufficiently far from the quencher.
- Cycle PCR and see how many cycls it takes to reach above threshold.
- if lots of gene copies, less cycles needed
Mutant K-ras as a biomarker for EGFR-targetted therapies
- used in colon cancer
- K-ras mutation products for resistance to EGFR-targetted therapies can be
- measured by real-time PCR
- ex. cetuximab blocks EGFR but downstream K-ras is constitutively active
- if they are k-RAS wt, then cetuximab will work (better than supportive care
What is a POC test? adv? disadv?
- test performed at or near the site where clinical care is delivered
- may be performed by patient or a HCP
- portable and convenient, rapid results, small sample volumes, no sample
- processing and decreased administrative documentation, ease of use, improved
- patient management
- quality is operator-dependent, operators are clinically focussed and not
- lab-trained, over utilization of testing and inappropriate use
- difficult to assure regulatory compliance
- sometimes not quantitative
- may increase costs
- increased risk of error
Explain glucose monitoring in diabetes
Explain glucose-monitoring test-strips
- test strips contain semi-permeable membrane to seperate 'plasma-like' liquid from
- cells as well as all the reagents needed to initiate a chemical rxn.
- electrodes are printed onto the strip using an ink-jet printer that dispenses
- carbon, gold or paladium for working electrode and Ag/AGcl for refrence
What is calibration of Glucose test strips?
- coding is the slope and intercept of a plot of current for an individual lot of strips
- vs. glucose concentration measured by a reference method (i.e. use hexokinase
- to measure)
What is the hexokinase reference method?
Draw it out
What is the ISO 15197 standard?
- greater than and including 95% of glucose test results within +/- 15mg/dL of the
- reference method at concentrations less than <75mg/dL (4.2mmol) and within
- 20% at concentrations greater than 4.2mmol
What is the Clarke-Error Grid?
- action a patient may take depending on their BG reading, and their target BG.
- Zone A; clinically accurate
- B: benign over/under estimation
- c: unacceptable over/under estimation
- D: unacceptable failure to detect
- E: unacceptablie error
CDE are clinically significant errors.
Cleverchek had D errors 3% of the time
Peritoneal analysis of blood glucose may be flawed
- is metabolised to glucose and maltose by GDH, but not GOX systems
thus falsely elevated glucose readings
Sources of error in glucose analysis:
- (assumed to be between 25-55%)
- -oxygen in rbcs may compete w/ mediators for e-; high HCT limits diffusion of
- samples and reactants; inverse relationship between HCT and strip response
What is normal PT?How to calculate INR?
- INR= controls for differences in activity of tissue factor
- INR= (PTtest/PTnormal)^ISI
- ISI= international sensitivity index
- INR usually 0.9-1.3, but 2-3 in coumadin patients.
- test strips contain thromboplastin, phospholipids and a peptide substrate.
- activation of clotting cascade produces thrombin which cleaves the peptide
- substrate, releasing electrochemically active phenyldiamine, generating a
Time to generate current is proportional to INR
- test strip contains thromboplastin: activation of clotting cascade causes clotting of sample which impedes the current passing through the meter current is reduced when clot forms
- algorithm used to determine INR and PT
How does an immunochromatography POCT work?
- sample migrates along strip where it binds gold particles linked to
- antibody conjugates, then later to reagents that form a colour if the protein
- is present (test line)
- control line has antibodies that recognizze the gold particles to ensure test
- is working
Examples of immunochromatography POCT
- HCG for pregnancy
- cardiac troponin
- Streptococcus group A (95% specific in 5 mins)
- -causes rheumatic fever and peritonsillar abscesses
- -throat swab culture is reference standard
- -immunochromatography detects STREPA carbohydrate antigen
- Pneumococcal urinary antigen (for C.A.P.)
- -test detects C-polysaccharide common to all pneumococcal
- -results in 5 mins: 92
- sensitivity, 100% specificity
What are the different lipoproteins? How are they separated? Wht is the most prevalent cholesterol?
chylomicrons, VLDL, LDL, HDL
seperated by density using ultracentrifugation (chylomicrons are lowest density and biggest size)
- LDL= 62% (stores cholesterol in blood stream)
- HDL= 19% (regulates LDL storage and promotes excretion)
How to monitor cholesterol?
- 1. hydrolyse cholesterol esters with cholesteryl esterase to release free cholesterol
- 2. cholesterol -> [cholesterol oxidase] -> cholestenone + H2O2
- 3. H2O2 + dye -> [peroxidase] colour (measured at 500nm)
How to measure TG?
Tg+ H2O -> Fatty acid + glycerol
glycerol is released from TG, which goes through enzymatic rxns to generate peroxidase.
- H2O2+ dye -> [peroxidase] colour
note that there is 10-20mg/dL overestimation of TG due to presence of free glycerol in blood
Measuring LDL and HDL
- plasma sample is ultracentrifuged to seperate VLDL, LDL and HDL.
- total cholesterol, TG, and HDL is measured
- VLDL is estimated, then LDL is calculated
LDL= total cholesterol - HDL - TG/5 [ this is the VLDL estimate]
fraction of oxygen bound to HB compared ot total O2 binding capacity of Hb in blood
oxygenated Hb (O2HB) and deoxygenated Hb are measred based on different spectrophotometric absorbance.
fraction of oxyhemoglobin (FO2Hb) is also measured.
SO2= [O2HB]/ [O2HB]+ [HHB] * 100
How to measure pO2
O2+ 4e- + 2H2O -> 4OH-
- oxygen diffuses across a membrant one an electrolyte solution and is then reduced at a a platinum cathode.
- type of galvanic cell; results in a current flowing which can be used to quantify amount of O2.
saturation of peripheral O2
based on differential absorbance of red and infrared light by O2Hb and HHb
CO2+ H2O-> H2CO3 -> H+ + HCO3-
CO2 diffuses across meembrane, and forms carbonic ancid, which dissociated to H+ and HCO3-, which reduces the pH
this is essentailly a pH electrode with a membrane.
What would be the 3 components of a blood gas analyzer?
- Severinghaus electrode (pCO2)
- clark electrode (pO2)
- pH electrode (pH)
How to measure HCO3-
bicarbonate concentrations are calculated by the blood gas analyzer from pH and pCO2 values.
- pH= 6.1 + log [HCO3-/CO2]
[CO2]= 0.0306 mmol/L * pCO2
Where is the most iron in your body?
- hemoglobin (2-2.5)
- ferritin (1-2.5)
- myoglobin 130 mg
- transferrin 3-5 mg
- enzymes 8 mg
How to measure serum Fe?
Fe-proteins [H+]->Fe3+ -> [ascorbic acid] Fe2+ + Ferrozine -> absorbance @ 562 nm
How to measure TIBC and % saturation
How to measure transferrin and ferritin?
- TIBC is the amount of FE that could be bound by saturating transferring and other iron-binding proteins in the serum.
- Determined by adding Fe3+ to the serum, removing any excess fe3+ by precipitation with MgCO3, then quantifying the Fe in the supernatant as before.
% saturation= Total Fe (ug/dL)/TIBC (ug/dL) * 100%
- Transferrin and ferritin measured by ELISA
- actual # of protein, not saturation.