Pharmaceutics Final Flashcards
Diagnostic Tests
- Needed to evaluate health and
1. Diagnose disease in a patient
2. Decide on the most appropriate treatment and,
3. Monitor the effects of treatment
Sensitivity
The fraction of patients who have the disease that is correctly predicted by the test
Sensitivity = TP/(TP + FN)
Specificity
The fraction of patients who do not have the disease that is correctly predicted by the test
Specificity = TN/(TN + FP)
True Positive
Individuals who have the disease that are correctly classified by the test
False Positive
Individuals who do not have the disease that are incorrectly classified by the test
True Negative
Individuals who do not have the disease that are correctly classified by the test
False Negative
Individuals who have the disease that are incorrectly classified by the test
Dichotomous Test
- One of two options
- Ex: Positive vs. Negative
Continuous Test
Range of values with a threshold set for a reference population vs. disease
Continuous Test: Prostate Specific Antigen
-Depending on where we set the threshold, this will determine the number of TP, FP, TN, and FN and therefore the sensitivity and specificity of the test
Receiver Operating Characteristic Curves
X: 100-Specificity (false positive rate)
Y: Sensitivity (true positive rate)
Provides optimal region for high sensitivity and specificity in order to choose a threshold for continuous data
Predictive Values
Positive: PPV = TP/(TP + FP)
-Likelihood that a positive test result will indicate disease
Negative: NPV = TN (TN + FN)
-Likelihood that a negative test result will indicate no disease
These numbers are affected by prevalence of disease
- High prevalence increases PPV, decrease NPV
- Low prevalence decreases PPV, increases NPV
Acute Myocardial Infarction
-Changes in cardiac biomarkers (especially troponin) above the 99th percentile of the upper reference limit, together with evidence of ischemia
Evidence of Ischemia
- Ischemic symptoms
- ECG changes associated with ischemia
- Pathologic Q-waves on ECG
- Imaging tests showing new loss of viable myocardium or new regional wall motion abnormality
Cardiac Troponin Complex
- Cardiac Troponin-I (cTnI) is the inhibitory form
- Troponin-T (cTnT) is the tropomyosin-binding form
- Both are found in skeletal muscle
- AMI causes release of both into the plasma
Principle of Immunoassay (e.g., ELISA)
- Monoclonal antibodies (Ab1) specific for the analyte (Ag) are used to bind the analyte in a plasma or serum sample and extract it for measurement - known as primary antibodies
- Secondary antibodies (Ab2) that are usually polyclonal and carry a label are used to measure the amount of immune complexes formed
Sandwich or Direct Immunoassay
- 96 well plates made of 96 tiny plates
- Primary antibody is stuck to the bottom of the plate
- Add plasma and incubate
- Wash to make sure all that is left is primary antibodies stuck to protein of interest
Abbott AxSYM Assay for cTnI
- Anti-cTnI (mouse monoclonal antibody) coated onto microparticle
- cTnI in serum sticks to antibody
- Anti-TnI (Goat)-AlkPhos (secondary antibody) sticks to certain part of cTnI separate from the primary antibody to form 4-methylumbelliferyl phosphate, which fluoresces at 365 nm
Limitations of cTnI Assays
- No primary cTnI reference standard to calibrate different assays - each commercial assay uses their own reference material for calibration
- Definition of “normal” reference limits is unique for each commercial assay and depends on the sensitivity (e.g., high sensitivity assays have different limits)
- Results with one assay are not easily comparable to those using another assay - best to follow changes in cTnI for an individual patient over time
Sensitivity and Specificity of cTnI Assays
- Sensitivity 91.8%
- Specificity 92.4%
- Sensitivity increases from 65% to >90% as time from presentation exceeds 5 hours, but specificity remains >90% at all times
- Sensitivity increases because the longer you wait after someone has MI, the more troponin you have in the body
- Specificity stays the same because the longer you wait, someone who does not have MI, still does not have MI
Kinetics of cTnI Elevation following MI
- Reperfusion of the myocardium by angioplasty or thrombolytic agents causes a more rapid decline in cTnI levels but higher initial values
- cTnI replaced creatinine kinase muscle and brain subunits (CK-MB)
Acute cTnI Elevation in Absence of MI
- Myocardial trauma (e.g., ICD firings, biopsy, cardioversion)
- CHF
- Myocardial surgery
- Renal failure
- Critically ill patients
- Drug toxicity (e.g., CO2 poisoning)
- Myocarditis
- Sepsis (due to TNF release)
- Other conditions
Hypothyroidism
- Deficiency of TH secretion (T3 and T4)
- Occurs in 2-15% of the population
- Primary due to thyroid gland failure
- Central due to pituitary gland dysfunction (low TSH)
Hyperthyroidism
- Elevated TH secretion (T3 or T4)
- Grave’s disease occurs in 0.4% of population
- Primary due to overactive thyroid gland
- Central due to pituitary/hypothalamus dysfunction (high TSH)
TSH Assays
- TSH is a 28-30 kDa dimeric glycoprotein consisting of an alpha and beta subunit
- Most assays are “sandwich” type direct immunoassays
- Assays calibrated using TSH reference preparation (WHO 2nd IRP 80/558)
- Results expressed as milli-international units of biological activity per L of serum (mIU/L) based on a comparison to a bioassay for TSH
Biocheck TSH Enzyme Assay
- Immobilized anti-TSH monoclonal antibody incubated in serum
- Wells in MicroELISA plate and washed
- Add goat anti-TSH-horseradish peroxidase (HRP)
- Measure absorbance at 450 nm
Linearity of TSH Measurement
- Limitation of ELISA is non-linearity because of saturation
- Non-linearity due to insufficient primary anti-TSH antibody coated onto microELISA plate
Competition T4 Immunoassay
- T4 plasma competes with labeled T4 (*T4) for binding to a monoclonal antibody (Ab1).
- *T4-Ab1 complex is separated from any free *T4 and measured
- Signal (chemoluminescent substrate) obtained is plotted vs. concentration of unlabeled T4 for a series of known concentrations of T4
- Unknown T4 concentration in the plasma is determined measuring its signal and referring to standard curve
-RLU is measured unknown amount, which corresponds to the real T4 concentration
Liver Diseases
- Acute hepatitis
- Cholestasis (blockage of common bile duct)
- Chronic hepatitis
- Cirrhosis
- Hepatocellular carcinoma
Algorithm for Assessing Liver Dysfunction
Abnormal Liver Function Test –> AST/ALT >3xURL, ALP Hepatocellular disease –> normal albumin = acute hepatitis; decreased = chronic hepatitis
Abnormal Liver Function Test –> AST/ALT 2xURL –> Cholestatic disease –> normal albumin = acute; decreased = chronic
Aspartate Aminotransferase
Aspartate + 2-oxoglutarate –AST–> oxaloacetate + glutamate
- Produced in liver, but other places too
- Less specific than ALT
ASSAY: Coupled Enzyme Assay
Oxaloacetate –MD–> Malate converts NADH to NAD+
Measure decrease in abs at 340 nm
Alanine Aminotransferase
Alanine + 2-oxoglutarate –ALT–> pyruvate + glutamate
- Produced in the liver primarily
- More specific than AST
ASSAY: Coupled Enzyme Assay
Pyruvate –LD–> Lactate converts NADH to NAD+
Measure decrease in abs at 340 nm
Coupled Enzyme Assay
- Product of one reaction becomes the substrate of another, which can be measured
- Usually measuring NADH to NAD+ by measuring the decrease in absorbance at 340 nm (NADH)
- Michaelis-Menten allows us to know that as long as the substrate is in excess, the rate of creation is constant and proportional to the amount of enzyme present
Alkaline Phosphate
- Several isoforms including bone, intestinal, liver and kidney ALP
- Most originates in the liver and bone-
- Increased serum ALP associated with liver dysfunction or bone osteoblastic activity
- There is increased synthesis of ALP in response to cholestatic disease
ASSAY: Colourmetric Assay
-Add a colorless compound and ALP turns it into a yellow compound, which can be measured at 405 nm
Serum Albumin Assays
Colourmetric Assay
-Add bromocresol green or purple and measure absorbance at 610-620 nm
RI for Hepatic Dysfunction
- AST/ALT increase 10-40 fold
- ALT increases 10-12 fold
- Albumin can remain normal or decrease
Kidney Function Tests
- Proteinuria
- Serum Creatinine
- Creatinine Clearance
- Urine: Glucose, uric acid, hemoglobin, leukocytes, pH and specific gravity
- BUM (serum)
Proteinuria
- Passage of serum proteins into the urine by the kidney glomeruli
- Proteins eliminated in the urine should be
Serum Creatinine
Creatinine is synthesized in the kidneys, liver and pancreas and transported to muscle and brain where it is phosphorylated
- A small proportion of phosphocreatine is converted to creatinine and excreted by the kidneys
- Serum creatinine is a marker for renal function
ASSAY:
- Jaffe Reaction
- Coupled Enzyme Reaction involving creatininase that generates hydrogen peroxide,m which converts a substrate into a coloured product that can be measured
Jaffe Reaction
SCr Assay
Picric Acid + Creatinine –OH–> RED-ORANGE COMPLEX (picrate-creatinine complex)
-Measure absorbance at 490-500 nm
Technical Problems
- Not specific for creatinine
- Small interferences from protein, glucose, ascorbic acid, ketones, cephalosporins, bilirubin and others
- Some assays compensate by subtracting a fixed concentration from the SCr value
- Some assays overestimate SCr by 20%
- Enzyme-based assays are more specific but still have some interferences (e.g., bilirubin)
Creatinine Clearance and GFR
-Clearance: Volume of plasma that is completely cleared of a substance per unit of time
-Creatinine is cleared mainly by glomerular filtration, thus its clearance is an estimate of GFR
+Tubular secretion can cause errors (7-10%)
-Creatinine Clearance measurements require timed urine collections to measure excretion rate of creatinine
Equations for Converting SCr into GFR
- Cockcroft and Gault Equation
a. Age
b. Weight
c. Sex
d. SCr - Modification in Diet in Renal Disease (MDRD)
a. Age
b. SCr
c. Sex
d. Black
RI for Renal Dysfunction
- Urinary protein and SCr increase
- GFR decreases
Criteria for TDM
- Narrow TI
- Correlation between plasma/serum concentration and efficacy or toxicity
- Variability in inter-individual PK
- Liver or kidney dysfunction may effect elimination
- Non-linear PK - Absence of a biomarker of therapeutic response
- Potential drug interactions that could effect plasma/serum concentrations
Analytic Methods for TDM
- Immunoassays
- GC-MS, LC-MS
- HPLC-UV
- Ion-selective Electrodes
Immunoassays
- Radioimmunoassay
- Enzyme-Linked Immunosorbent Assay (ELISA)
- Enzyme-Multiplied Immunoassay (EMIT)
- Cloned-Enzyme Donor Immunoassay (CEDIA)
- Chemiluminescence Immunoassay
- Most common
- Easily automated
- Versatile
- Relative specific for drug
Radioimmunoassay
- Incubate antibody-bound radiolabeled drug affixed to plate + drug in plasma/serum
- Drug competes with radiolabeled drug
- Measure antibody-bound radiolabeled drug to determine amount of antibody-bound drug there is indirectly
Heterogenous because you have to wash away free radiolabeled drug
Ex. Digoxin
Digoxin RIA
Interfering substances
- Digoxin-like immunoreactive factor (DLIF)
- Present in pregnant women and neonates
- Affinity to primary antibody is much lower than actual drug, but can still interfere
- Different assays have more specificity - Other substances with steroid structure
EMIT Assay
Enzyme-Multiplied Immunoassay
- Antibody-bound drug-enzyme conjugate (inactive) + drug in plasma/serum
- Real drug competes with enzyme-bound
- Free drug-enzyme conjugate will react with substrate and be active for measurement
- Measure colour
Homogeneous because the bound drug-enzye conjugate is inactive and so no separation is needed
Ex. Gentamicin
EMIT 2000 Gentamicin Assay
EMIT Beckman-Coulter
Drug: Gentamicin
Enzyme: G6PD
Substrate: Glucose
Conversion of glucose to gluconolactone by G6PD results in production of NADPH, which is measured at 340 nm
Chemilluminescence
Acridinium esters undergo a chemical reaction when exposed to hydrogen peroxide that produces light that can be measured
Ex. Phenytoin
Abbott Architect Phenytoin CIA
- Phenytoin-Acridinium conjugate bound to antibody on micro particle + phenytoin in plasma/serum
- Measure proportion of bound forms indirectly by measuring chemiluminescence for phenytoin-acridinium conjugate bound to micro particle
Heterogeneous immunoassay
Clinical Toxicology
- ALCOHOL
- SALICYLATES
- ACETAMINOPHEN
- Amphetamines
- Cannabinoids
- Cocaine
- Opiates
- PCP
- Sedative hypnotics
- Carbon monoxide
Clinical Toxicology: Patient Considerations
Primary survey of patient condition
- Patient history
- Initial physical exam
- Ancillary tests
Secondary survey of patient condition
- Complete and more detailed physical exam
- Exam of items brought with patient
- More specific lab tests
Clinical Toxicology: Point of Care Drug Screen
- Drug in urine competes for binding with fluorescently labeled drug to channel in cartridge
- Fluoresence measured in POC meter
- Only tells presence of drug over threshold, not actual concentration
Limitations
- Drug may be present in lower than threshold conc
- Does not indicate if level of drug is toxic, only that drug is present in urine
- Does not provide concentration of drug
Clinical Toxicology: Ethanol Assay
- Enzymatic Assay
Ethanol + NAD+ –ADH–> Acetaldehyde + NADH (measured at 340 nm) - Osmolar Gap
- Measure freezing point depression of plasma sample in osmometer
- Osmolar gap = measured osmolality - calculated osmolality (which is about 275-295 mOsm/kg for plasma)
- Serum osmolality increases by 10 mOsm/kg for each 0.06 g/100 mL ethanol
Note: Other alcohols can be measured using GC
Clinical Toxicology: Acetaminophen Assay
Enzymatic
Acet –acidamide amidohydrogenase–> p-aminophenol –o-cresol; NH3/Cu2+–> BLUE COLOUR
Clinical Toxicology: Acetaminophen Rumack-Mathew Nomogram
- Hepatic toxicity of acetaminophen due to reactive metabolite (NAPQI)
- Probability of hepatic toxicity depends on the time since ingestion and the plasma concentration of acetaminophen defined by nomogram
- Y-axis: Acet plasma conc; X-axis: Time since ingestion
- There is a 4 hour delay between ingestion and assessment of clinical significance
NOTE: Antidote is acetylcysteine
Clinical Toxicology: Salicylate Assay
- Trinder Reaction (Colourmetric)
Aspirin + Fe3+ –> VIOLET (measure at 540 nm) - Enzymatic Assay
Salicylate + NADH + H+ –salicylate hydroxylase–> Catechol + NAD+
Measure decrease in abs at 340 nm
Biomaker
Characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic response to a therapeutic intervention
Role of Biomarkers in Cancer
- Screening for early detection
- Aiding in diagnosis of cancer
- Predicting survival from cancer
- Predicting therapeutic response
- Tumour staging
- Detecting recurrence of cancer
- Monitoring the effectiveness of treatment
Examples of Biomarkers in Cancer
- CEA –> colon –> IA, IHC
- PSA –> prostate –> IA
- CA125 –> ovarian –> IA
- CA19-9 –> pancreatic –> IA
- HER2 –> breast, gastric –> IHC, FISH
- EGFR –> colon, lung –> IHC
- ER, PgR –> breast –> IHC
- Kras –> lung –> RT-PCR
- Bcr-Abl –> CML –> RT-PCR
- CD20 –> B-cell lymphoma –> IHC, FC
Carcinoembryonic Antigen
CEA - colon cancer - IA, IHC
- Cell surface glycoprotein adhesion molecule
- Elevated in the serum in COLORECTAL, lung, gastric, breast and pancreatic cancer but some false positives and negatives
- Serum CEA decreases with successful treatment of colorectal cancer and rises with recurrence
USED for detection of Recurrent COLORECTAL CANCER
-At >10 U/L, sensitivity is 50%, specificity is 99%; PPV is 74%, NPV is 92%
Prostate Specific Antigen
PSA - prostate cancer - IA
- Glycoprotein protease found in prostate gland
- Elevated in prostate cancer
- Used as a screening tool for prostate cancer, followed up by biopsy for diagnosis
CA125
ovarian cancer - IA
- Glycoprotein expressed mainly by ovarian cancer but also endometrial, pancreatic, lung, breast and colorectal cancer
- Increased in serum as disease progresses from Stage I
- At >35 U/L, sensitivity is 95% and specificity is 82%
- Detection of recurrent or residual disease after treatment but NOT for screening except in high risk women
CA19-9
pancreatic - IA
- Glycolipid secreted by normal pancreas and biliary duct cells
- Elevated in pancreatic cancer, gastric, hepatobiliary and other cancers
- At >37 U/mL, sensitivity is 69-93%, specificity is 76-99%
HER2
- Overexpressed in 20-25% of breast cancers
- Target for trastuzumab (Herceptin)
- Patients selected for treatment by IHC staining for HER2 or by FISH
Immunohistochemistry (for HER2)
- Add primary antibody that recognizes HER2 and wash
- Add secondary antibody conjugated to an enzyme (HRP)
- Add DAB substrate and hydrogen peroxide - forms a colour (brown) by reacting with enzyme on the tissue section
Fluorescence in Situ Hybridization (for HER2)
- Incubate with fluorescently labeled oligonucleotides that bind to copies of HER2 gene
- Examine cells for fluorescence - pink is HER2, green is control
Estrogen and Progesterone Receptors
- Both are measured in breast cancer by IHC staining of a tumour biopsy
- Positive staining of more than 1% of tumour cell nuclei is considered ER-positive, but response to anti-estrogen therapy is most common with positive staining of over 20% of cells
- Can use tamoxifen and aromatase inhibitors (e.g., Letrozole) to treat ER positive breast cancer
Mammaprint
- Probes 70 genes to separate early stage node-negative breast cancer patients into low or high risk for recurrence
- Patients at high risk may need chemo
OncoType DX
- Probes 21 genes to derive a recurrence score for ER-positive early stage breast cancer
- Aids in decision-making about treatment
- Performed by Reverse-Transcriptase PCR
CD20
Non-Hodgkin’s Lymphoma
- Cell surface phosphoprotein expressed on normal B cells and on non-Hodgkin’s B-cell lymphoma
- Target for monoclonal antibody, rituximab (Rituxan)
Flow Cytometry analysis of CD20 expression on B-cells
- Based on the binding of fluorescently-labeled antibodies to cells and their sorting by fluorescence intensity
- More than 90% of B-cell lymphomas express CD20
Bcr-Abl
Translocation in CML
- Translocation of Abl gene from chromosome 9 to 22 in 90% of cases
- Forms Bcr-Abl fusion gene that leads to protein that is constitutively active (tyrosine kinase) and responsible for proliferation of CML
- Imatinib (Gleevec) is a tyrosine kinase inhibitor which competes with ATP for the phosphorylation sites on Bcr-Abl protein
- Can use real time RT-PCR to measure Bcr-Abl transcripts using threshold value
K-ras
Biomarker for EGFR-Targeted Therapies
- Mutation in K-ras predicts resistance for EGFR-targeted therapies (e.g., cetuximab or panitumumab)
- Can be measured using real time PCR
- Mutation leads to permanently active EGFR signaling and continue proliferation; treatment works upstream of this and so is useless when the mutation is present
Point-of-Care Tests
- Lab testing performed at or near the site where clinical care is delivered
- May be performed by the patient or by a health care professional - decreases time required for a lab result allowing adjustments in patient care to improve outcomes
- Many different analytes
POCT Pro and Con
PRO
- Portable and convenient
- Rapid results
- Small sample volumes
- No sample processing and decreased administrative documentation
- Specific identification of marker
- Ease of use
- Improved patient management
CON
- Quality is operator-dependent
- Operators are clinically-focused and not lab trained
- Overutilization of testing and inappropriate use
- Difficult to assure regulatory compliance
- Sometimes not quantitative
- May increase costs
- Increased risk of error
Glucose Monitoring in Diabetes
- Target glucose concentration: 90-130 mg/dL (5.0-7.2 mmol/L)
- Prolonged hyperglycemia increases the risk for diabetic retinopathy and cardiac disease as well as tissue infections (sometimes leading to amputations)
- Hypoglycemia is also a serious risk that can lead to coma and death
- Blood glucose monitoring is commonly employed in insulin-dependent Type I and II diabetics to maintain levels within acceptable range by insulin administration
Blood Glucose Measurement - GOx and GDH
SEE PICTURES
Blood Glucose Measurement - Mediators
- Mediators receive and transfer electrons from GOX or GDH driving the reaction and producing the electrons we can measure once glucose is converted
- Ferricyanide, ferrocene, PQQ
Glucose Monitors
- Electrochemical Cell
- Oxidation occurs at the anode, measuring actual reaction where the mediator is producing electrons upon glucose conversion
- Reduction occurs at the cathode, measuring reaction that is constantly occurring in the environment as a control
Glucose Test Strips
- Strip contains semi-permeable membrane to separate “plasma-like” liquid from cells as well as all the reagents (e.g., mediators and enzymes) needed to initiate the electrochemical reaction
- Electrodes are printed onto the strip using an ink jet printer that dispenses carbon, gold or palladium for working electrode and Ag/AgCl for counter/reference electrode
Test Strip Calibration
-Coding is the slope and intercept of a plot of current (uAmps) for an individual lot of strips vs. glucose concentration measured by a reference method
-This differs for each lot of strips and previously needed to be entered into the glucometer
-Reference method uses hexokinase to measure glucose
+SEE PICTURES
+Uses hexokinase as the enzyme and converts glucose + ATP into gluc-6-phosphate –> converted to 6-phsophogluconate with the conversion of NADPH to NADP+ (at 340 nm)
Commercial Glucometers
- One Touch Ultra - GOx, ferricyanide
- Ascensia Contour - GDH, ferricyanide
- Accucheck Aviva - GDH, PQQ
ISO 15197 Standard - Health Canada
In 2010, they determined that a lot of meters were not compliant with ISO 15197 standard
Clarke Error Grid
- Used to figure out if glucose meters are good
- The dots are glucose measurements and the Y-axis is the concentration according to the meter, X is glucose concentration according to lab tests (“real”)
- We want a perfectly diagonal line for perfect meter
ZONES
- A: Clinically accurate
- B: Benign over/under-estimation; still compliant
- C: Unacceptable over/under-estimation
- D: Unacceptable failure to detect; meter telling you that you have high levels when you don’t or low when they are high
- E: Unacceptable errors
Icodextrin in Peritoneal Dialysis
-Metabolized into glucose and maltose
-Cannot be differentiated from glucose by GDH, better to use GOx
-Causes falsely-elevated glucose readings
-US FDA Warning
+13 deaths from 1997-2009 in patients; 10 who were receiving peritoneal dialysis with icodextrin solutions and 3 received other maltose containing drugs
+Falsely-elevated glucose readings specific for glucometers using GDH-PQQ test strips
Other Drug Interferences of Glucose Monitoring
Drug –> Interfering Concentration (mg/dL)
- Acetaminophen –> >20
- Salicylic acid –> >50
- Tetracycline –> >4
- Dopamine –> >13
- Ephedrine –> >10
- Ibuprofen –> >40
- L-DOPA –> >5
- Methyl-DOPA –> >2.5
- Tolazamide –> >100
- Ascorbic acid –> >3
Sources of Error in Glucose Measurement
- Hematocrit - should be 25-55%
- Oxygen in RBCs may compete with mediator for e-
- High Hct limits diffusion of sample and reactants
- Inverse relationship between Hct and strip response - Skin Contamination
- Glucose on fingertips can cause false high readings (e.g., grapes)
- Always wash hands before collecting blood - Miscoding
- Correct coding must be entered for calibration of the glucometer and accurate glucose reading
First Response Pregnancy Test
- Remove overcap and expose the absorbent tip to urine. Wait for 1 minute.
- Observe for one pink line (not pregnant) or two pink lines (pregnant).
- Measures increased human chorionic gonadotropin (hCG > 25-50 IU/L)
Immunochromatography POCT
- Sample migrates along the strip where it binds to gold particles linked to antibody conjugates and then later to reagents that form a colour if the protein is present (test line)
- The control line has antibodies that recognize the gold particles to ensure the test is operating correctly
Cardiac Troponin-T POCT
- Immunochromatography
- Blood travels through the test and picks up cardiac troponin to indicate heart disease
- Gold anti-TnT
- Biotin anti-gold (TnT) complex
- Streptavidin-Label (test)
- TnT-Label (control)
Streptococcus Group A POCT
- Immunochromatography test using throat swab detects Strep A carbohydrate antigen (95% specific in 5 minutes)
- Strep A causes rheumatic fever and peritonsillar abscesses
- Throat swab blood agar plate culture is reference standard
Pneumococcal Urinary Antigen POCT
- Immunochromatography detects C-polysaccharide common to all pneumococcal serotypes (92% sensitive, 100% specific in 15 minutes)
- CAP most frequently caused by S. pneumo
Lipoproteins
- Molecules made up of lipids and proteins
- Proteins act as emulsifying agents so that the lipid can travel through the body
- Separated by density using ultracentrifugation into chylomicrons, VLDL, LDL and HDL
- LDL is 62% cholesterol, HDL is 19%
- LDL stores cholesterol in the blood stream
- HDL regulates LDL storage and promotes excretion
Risk Factors for Coronary Heart Disease
- Age > 45 for men, 55 for women
- Family history
- Cigarette smoking
- Hypertension
- Diabetes
- High cholesterol (esp LDL)
CHD and Cholesterol
-Risk for death due to CHD is 4x higher at a total cholesterol level of 300 mg/dL (7.2 mmol/L) than at 150 mg/dL (3.6 mmol/L)
Classification of Cholesterol Levels
- LDL: 4.1-4.9 mmol/L is HIGH
- Total Cholesterol: >6.2 mmol/L is HIGH
Target Cholesterol Levels and CHD
LDL Goals
-CHD and CHD 10 year risk > 20% –>
Measurement of Cholesterol
Enzymatic Assay
- Hydrolyse cholesterol esters with cholesteryl esterase to release free cholesterol
- Cholesterol –cholesterol oxidase–> cholestenone + H2O2
- H2O2 + dye –peroxidase–> colour (500 nm)
Measurement of Triglycerides
- Enzymatic Assay
- Glycerol is part of the chain, but there is a lot of glycerol present in the blood, which causes an overestimation of the triglycerides in the body by 10-20 mg/dL
Measurement of LDL and HDL
-Plasma same is ultracentrifuged to separate VLDL, LDL and HDL
-Total cholesterol, TG and HDL are measured
-VLDL is estimated (TG/5) and LDL is then calculated as:
LDL = TC - HDL - TG/5
Arterial Blood Gases
- Oxygen
- Oxygen saturation (SO2)
- Fraction oxyhemoglobin (FO2Hb)
- Partial pressure of oxygen in plasma (pO2)
- Pulse oximetry (SpO2) - Carbon Dioxide
- Expressed as partial pressure CO2 in plasma (pCO2)
Oxygen Saturation
- SO2 is the fraction of O2 bound to hemoglobin compared to total O2 binding capacity of Hb in blood
- Oxygenated Hb (O2Hb) and deoxygenated Hb (HHb) are measured based on different spectrophotometric absorbance
- Fraction of oxyhemoglobin (FO2Hb) is also measured
SO2 = [O2Hb]/([O2Hb]+[HHb]) x 100%
- Normal levels should be 95-100%
- Below 90% is hypoxic
- Below 80% can lead to compromised organ function
Pulse Oximetry
- Measures absorbance of oxyhemoglobin and hemoglobin and calculates ratio
- Based on differential absorbance of red (660 nm) and infrared (910 nm) light by O2Hb and HHb
- Estimates oxygen saturation (SpO2)
pO2 and pCO2
- At sea level, barometric pressure is 760 mmHg
- pO2 = 760 x 20.93% = 159 mmHg
- pCO2 = 760 x 0.03% = 0.23 mmHg
-In blood and tissues, there are changes in pO2 and pCO2 in the blood from arterial blood in the lungs to venous blood returning from the tissues
+pO2: Arterial > Venous
+pCO2: Venous > Arterial
-Most oxygen in the blood is carried by Hb (98%)
+pO2 and pCO2 only reflect dissolved O2 and CO2 in the blood
-pO2 gives idea of how effective the lungs are at pulling oxygen from the atmosphere to blood
Measurement of pO2
Clarke Electrode
- Oxygen diffuses across a membrane into an electrolyte solution and is then reduced at a platinum cathode
- Type of galvanic cell –> results in a CURRENT flowing which can be used to quantify amount of O2
O2 + 4 e- + 2 H2O –> 4 OH-
Measurement of pCO2
Severinghaus Electrode
- CO2 diffuses across a membrane and forms carbonic acid, which then dissociates to H+ and HCO3- –> reduced pH
- Based on reduction in pH, CO2 can be determined
Blood Gas Analyzer
-Includes all 3 electrodes to measure pH, pO2 and pCO2
Iron in the Body
- Hemoglobin
- O2, CO2 and iron transport - Myoglobin
- Hb in muscle - Transferrin
- Iron transfer - Ferritin
- Iron storage and release in controlled fashion - Cytochromes and other enzymes
- Iron binding and various other functions
Laboratory Iron Markers
Important Measurements
- Serum Fe
- Transferrin
- Ferritin
- %Fe Saturation
- TIBC (total iron binding capacity; measure of how well transferrin is working)
Measurement of Serum Fe
Fe-Proteins –[H+]–> Fe3+ –ascorbic acid–> Fe2+ + Ferrozine –> absorbance at 562 nm
Changes in Iron Markers in Disease
- Iron deficiency
- Decrease in serum iron, %saturation and ferritin
- Increase in transferrin - Chronic anemia
- Decrease in serum iron and %saturation
- Decrease or same transferrin
- Increase or same ferritin - Iron Poisoning
- Decrease in transferrin
- Increase in serum iron, %saturation and ferritin
NOTE: transferrin and ferritin are measured by ELISA
