Analytical Flashcards
What is the gold standard method for separating MacroPrl and Prolactin?
Gel filtration chromatography
What are the mechanisms by which lipaemia causes interference? (name 4)
- Spectrophotometric interference (light absorption and light scattering)
- Volume depletion effect (e.g. pseudohyponatreamia)
- Partitioning of the sample
- Physicochemical mechanisms (e.g., disturbance of the electrophoretic pattern).
Methods for removing lipids from a lipaemic sample?
- Ultracentrifugation or high speed centrifugation
- Lipid clearing agents such as Lipoclear
Reaction catalysed by CK used in enzymatic method?
Creatine phosphate + ADP –> Creatine + ATP
In the enzymatic spectrophotometric method of bicarbonate measurement, PEPC catalyses the reaction of HCO3- + PEP to form ___ ?
Oxaloacetic acid
The enzyme PEPC is involved in the spectrophotometric method of bicarbonate measurement. What does PEPC stand for?
Phosphoenolpyruvate carboxylase
Approximately how much CO2 is lost from uncapped tubes per hour?
4mmol/L
What can interfere with the paracetamol enzymatic assay?
N-acetylcysteine
Salicyclic acid
What is the method used for paracetamol measurement?
Enzymatic/colourimetric using aryl acylamidiase
What issue can occur with creatinine measurement in the setting of paracetamol OD?
Therapeutic concentrations of NAC can interfere with the Cr enzymatic method
Method for ethanol measurement?
Enzymatic method using alcohol dehydrogenase
In scanning spectroscopy in investigating for CSF bilirubin, at what wavelengths do the peaks for oxyhaemoglobin and bilirubin occur?
Oxyhaemoglobin peak 415nm
bilirubin peak 476nm
What are the different fractions of bilirubin
Unconjugated (alpha)
Conjugated: mono (beta) and di (gamma)
Delta bilirubin: covalently bound to albumin
Reference method for bilirubin measurement
Modified Jendrassik and Grof
- Caffeine-benzoate-acetate accelerator
Name 6 methods for bilirubin analysis
- Chemical (diazo reaction)
- Direct spectrophotometry
- Enzymatic (oxidation)
- HPLC
- Scanning spectrophotometry (CSF)
- Reflectance (transcutaenous bilirubin)
What can cause interference in Diazo reaction for bilirubin measurement
Haemolysis
Lipaemia
Paraproteinaemia
What is the pKa of bicarbonate?
6.1
What will happen to the pH of sample if the specimen is left uncapped?
pH will increase
Loss of CO2 from sample into room air
What is the difference between actual and standard bicarbonate measurements on blood gas machine?
Actual bicarbonate is a derived measurement calculated from pH and pCO2 on anaerobically collected sample.
Standard bicarbonate indicates bicarbonate level at 37C and pCO2 of 40mmHg
Osmolality is a _______ property of solutions that depends on the number of dissolved particles present in the solution
Colligative
As the number of dissolved particles in a solution increases, the freezing point and vapour pressure of a solution will ________
decrease
Formula for calculating urine osmolality
= 2 x (Na + K) + urea
Interferences in osmolality measurement
Citrate can increased measured osmolality
Any particular matter (e.g. microclots in serum/plasma)
Interferences in analytical measurement of iron?
Chelators (e.g. EDTA, citrate)
What is osmolality?
The concentration of a solution expressed as the total number of solute particles per litre.
What are colligative properties?
The physical changes that result from adding solute to a solvent.
The Jaffe method involves Creatining reacting with ________ in alkaline medium to produce an orange-red complex?
Picrate
Sources of interference in the Kinetic Jaffe method?
Ketoacids
Cephalosporins
Bilirubin - negative interference
The kinetic Jaffe method measures creatinine-picrate at what wavelength?
500nm
What enzymes are involved in the enzymatic method for Creatinine (Abbott Alinity)?
- Creatininase
- Creatinase
- Sarcosine oxidase
In the enzymatic method for creatinine, detection of ______ is measured at 548nm?
H2O2
Causes of positive inteference in enzymatic assay for Creatinine?
Lidocaine
Creatine supplements
Causes of false low results of Creatinine with Alinity enzymatic assay?
N-acetyl-L-cysteine
Alpha-methyldopa
Interferences in Jaffe method?
Positive bias from:
alpha-ketoacids
Cephalosporins
Ascorbic acid
Glucose
Glutathione
Uric acids
Reference method for Creatinine
Isotope Dilution Mass Spectometry
What is Stokes shift? (Fluorometry)
The difference between the maximum wavelength of the excitation light and the maximum wavelength of the emitted fluorescence
Do faecal elastase assays detect the porcine elastase used in supplements?
No
What is the type of test used for FOBT?
Immunochemical test for human globin
(iFOB uses turbidimetry)
Components of a Fluorometer
- Excitation source
- An excitation monochromator
- Cuvet
- Emission monochomator
- Detector
Excitation sources that can be used in Fluorometers
Xenon lamp
Lasers
Light-emitting diode (LEDs)
The absorption spectra of most fluorescent compounds of interest are in the spectral region of…
300-700nm
Types of photodetectors used in Fluorometry
Photomultiplier tubes (PMT)
Charge coupled detectors (CCD)
Patient preparation for a Hydrogen/Methane test?
- No antibiotics for 4 weeks prior
- Avoid fermentable carbohydrates day prior
- Overnight fast
- No smoking
- Oral hygiene (if poor may influence results)
How are Hydrogen and Methane analysed following a breath test?
chromatographic separation of gases on
molecular sieve column
Gold standard test for determining Small Intestinal Bacterial Overgrowth (SIBO)
Culture of intestinal fluid
Breath tests are surrogate markers
Causes of false negatives in a urea breath test for H. pylori
PPIs (past 2 weeks)
Antibiotics (past 4 weeks)
Bismuth therapy (past 4 weeks)
Active peptic ulcer bleeding
Recommended method for metanephrine measurement
LCMS
Pre-analytical factors influencing metanephrine results
- Position (lower reading with supine)
- Recent intake catecholamine rich foods (banana, pineapple, walnuts) - increase in 3MT
- coffee - increase normet
- exercise - can increase normet, met
- medications e.g. antidepressants, sympathomimetics
- stress, critical illness
Method used in NIPT
Next generation sequencing
Difference between Rayleigh and Raman light scattering
Rayleigh scattering occurs with no change in wavelength
Raman scattering occurs with lengthening of a wavelength
How is phosphorescence different to fluorescence?
- results from relaxatin of molecules in an excited triplet electronic state
- decay time is longer
- shows a larger shift in emission light wavelength
What does MLPA stand for
Multiplex Ligation dependent Probe Amplification
6 steps of MLPA
- Denaturation
- Hybridisation
- Ligation
- Amplification by PCR
- Fragment separation
- Data analysis
Genetic analysis technique commonly used for imprinting syndromes (e.g. Angelman, Prader-willi)
MS-MLPA
Methylation specific MLPA
A key difference between 2nd and 3rd generation PTH assays is that 2nd gen assays are affected by cross reactivity with … ?
C-terminal fragments
Components of Atomic Absorption Spectrophotometer (flame)
- Hollow cathode lamp
- Chopper
- Flame
- Monochromator
- Detector
Principle of AAS
Each element absorbs light of a specific (unique) wavelength
What is HbA1c
The fraction of haemoglobin where a glucose has been nonenzymatically attached to the N-terminal valine of the beta chain
In agarose gel electrophoresis, most proteins migrate towards the …
+ve anode
In capillary zone electrophoresis, proteins migrate towards the…
cathode (-ve)
Order of detection of proteins in CZE
gamma; B-2; B-1; a2; a1; albumin
opposite to gel electrophoresis
Conditions other than myeloma/MGUS that can produce monoclonal band on EPP?
- Plasmacytoma
- Lymphoma (including Waldenström’s
macroglobulinaemia) - Amyloidosis
- Cryoglobulinaemia
- Chronic lymphocytic leukaemia
Changes seen on electrophoresis with nephrotic syndrome?
Hypoalbuminaemia
Lipoproteins
Alpha2 macroglobulin
Hypogamma
How does glucose concentration differ in whole blood, plasma and serum?
Whole blood <plasma < Serum
(serum 2-5% higher than plasma due to fluid shifts from erythrocytes to plasma because of anticoagulants)
Requirements (definition) of a high sensitivity troponin assay
- CV <10% at 99th percentile URL
- detectable cTN in at least 50% of “normal” male and female patient populations
- LoQ <20% CV
Rerpoted in ng/L
What does FTIR stand for?
Fourier Transform Infra-Red Spectrometer
Components of HPLC
- Solvents
- Pump
- Column
- Sample injector
- Detector
Reverse phase chromatography has a stationary phase that is …
Non polar
Commonly used solvents in reverse phase chromatography
Water
Methanol
Acetonitrile
What is used as the stationary phase in ion exchange chromatography
Resin
Mobile phase: aqueous soln of salt + buffer
What methods are used in the lab to purify water?
- Filtration
- Reverse osmosis
- Deionisation
- UV oxidation
Water that is fit for most laboratory purposes is termed
Clinical Laboratory Reagent Water (CLRW)
Specifications of CLRW
- Microbiological content <10 conlony forming units/mL
- Resistance at 25degC >10 MOhm
- Particulate matter (water passed through 0.2 um filter)
- Total organic content **<500 ng/g
In the enzymatic method of total bile acids, the rate of formation of __ ? __ is determined by measuring the change of absorbance at 405nm as bile acids are oxidised by 3-a-HSD.
Thio-NADH
Interferences in the enzymatic method for bile acids?
- Haemolysis
- Lipaemia
- Ursodeoxycholic acid (UDCA) – patients on this therapy are not suitable for analysis.
The direct method for bilirubin measures which fraction of bilirubin?
Conjugated
In the direct diazo method, bilirubin couples with a diazonium salt in the presence of saulfamic acid to form the coloured compound…
Azobilirubin
The diazo method for bilirubin measures the increase in absorbance due to azobilirubin at what wavelength?
548nm
What is used as the accelerator in the Jendrassik-Grof method for bilirubin measurement?
Caffeine
Modification of this method includes the addition of surfactant
Reference method for ALP uses what as the substrate?
4-nitrophenyl phosphate
Kinetic spectrophotometry
Buffer = AMP at 37C
Most heat stable form of ALP
Placental ALP
Methods of measuring bone specific ALP
- Colourimetric assays + pre treatment to select BALP
- Immunoassays
Misleading causes of NAGMA
- Bias and imprecision in Na+, K+, Cl-, HCO3-
- low albumin
- high cations e.g. Ca2+, monoclonal proteins
- Interference with chloride electrode - e.g. bromide
Measurement issues with urine bicarbonate
- rapidly lost to the air in the form of CO2
- concentration too low to measure on enzymatic automated analysers
- frequently done on blood gas analysers - ?validation
The Friedewald equation is inaccurate when:
- when TG are high
- when LDL is low
- in patients with type 3 hyperlipoproteinaemia
In a K+ ISE, potassium selective liquid membrane uses…
Valinomycin
The calcium ISE is a liquid membrane electrode. It uses an ion-selective carrier such as..
Dioctylphenyl phosphonate
Reference electrode commonly used in pH electrode
Ag/AgCl immersed in KCl
Ion selective electrodes can be classified into 4 main types:
Based on selective membrane used
- Glass
- Solid state/crystalline
- Liquid ion-exchange
- Enzyme
In a pO2 electrode, the anode consists of..
Ag rod coated with AgCl
Oxidatio of Ag occurs
In a pO2 electrode, the cathode consists of…
Platinum wire encased in glass
Reduction of O2 occurs here
What is the role of the platinum black catalyst in the pO2 electrode?
Conversion of H2O2 to O2 for reduction at the cathode
H2O2 is produced from O2 not completely reduced.
In blood gas measurement of glucose/lactate, what occurs in the middle enzyme layer?
Production of H2O2
Glucose + O2 -> Gluconic acid + H2O2
Lactate + O2 -> Pyruvate + H2O2
Lipaemic index is measured at what wavelengths
660/700nm
At which there is only a small influence by the haemolysis + icterus
Haemolysis index is measured at what wavelengths
570/600nm
To repress the influence of the icteric content as much as possible
Icteric content is measured at what wavelength
480 / 550 nm
Analytes which are higher in serum than plasma
- Lactate
- Troponin
- K+
- P
- Glucose
- ALP
- Albumin
Analytes which are higher in plasma than serum
- LDH
- Protein (due to Fibrinogen)
Lipaemic samples can result in falsely low values due to increased turbidity. Which analytes?
ceruloplasmin, prealbumin, transferrin
measured by immunoturbidimetry
Steps of solid phase extraction
- Conditioning - Solvent is added to cartrigde containing silica
2.** Load **sample - **Wash **- potential interferences remove
- Selective elution of analyte for further work up/analysis
In the routine method for bilirubin, bilirubin couples with a ________
Diazo reagent
Interferences in Diazo method for Bilirubin
- Haemolysis
- Lipaemia
- Indicans (present with high concentrations of ureaemia)
- Indocyanine green
How does haemolysis cause interference in Diazo method for bilirubin?
- Spectral interference and
- Free Hb inhibits the reaction
Possible acclerators in Diazo reaction
Methanol
Caffeine (Jendrassik-Grof)
Dimethyl Sulfoxide
Surfactant
Enzyme reactions used in ALT method
- Alanine + alpha-ketoglutarate -> Pyruvate + Glutamate
- Pyruvate + NADH -> Lactate + NAD+
Measurement at 34onm
Enzyme reactions in AST method
- Asparate + alpha ketoglutarate -> Oxaloacetate + Glutamate
- Oxaloacetate + NADH -> Malate + NAD+
Malate dehydrogenase catalyses 2nd reaction
Method for LDH
Lactate + NAD+ -> Pyruvate + NADH + H+
IFCC recommended forward reaction
Preferred speciment for LDH
Serum
Plasma may be contaminated with platelets, high conc of LDH
Enzyme reactions used in measurement of CK
- Creatine phosphate + ADP -> Creatine + ATP
- ATP + glucose – (Hexokinase)–> Glucose-6-phosphate + ADP
- G-6-P + NADP –(G6PD)–>6-P-G + NADPH + H
These reactions occur in presence of NAC (Enzyme reactivator)
On a CSF xanthochromia scan, net bilirubin absorbance and net oxyhaemoglobin absorbance are measured at what wavelengths?
Net Bilirubin Absorbance: 476 nm
Net OxyHaemoglobin Absorbance: 415nm
A CSF xanthochromia scan result is consistent with SAH if net bilirubin absorbance (NBA) is…
> 0.007 AU
(not the only criteria. Also NOA >0.02 with vis peak or CSF protein <1)
What is the significance of a CSF total protein >1g/L in interpreting a xanthochromia scan?
Interpret with caution as ↑ CSF protein → ↑ CSF bilirubin
CSF bilirubin `correction’ NOT recommended if protein CSF > 1 g/L
What is an ionophore?
Chemical species that reversibly binds ions
Used in ion selective electrodes
How do activity coefficients of ions change with concentration?
Diluted samples e.g calibrators activity coefficient ≈ 1 (more stable)
Concentrated specimens e.g. Blood, serum - Activity coefficient
varies and usually <0.7
Analytical method for Beta-hydroxybutyrate
Beta hydroxybutyrate dehydrogenase +/- electron mediator
Routine method for total cholesterol - what are the 3 enzymes used?
- Cholesterol esterase
- Cholesterol Oxidase
- Peroxidase
H2O2 reacts to form chromophore (quinoneimine dye) - measured at 500 nm
Interferences in Total Cholesterol enzymatic method
- N-Acetyl-L-Cysteine - may lead to falsely low results
- Bilirubin – negative interference
- Ascorbic acid - negative interference
What are the enzymes used in the measurement of triglycerides?
- Lipase (+protease)
- Glycerol kinase
- Glycerol phosphate oxidase
H2O2 formed - reacts to form a coloured dye
High glycerol levels in blood can occur with
- increased endogenous glycerol in liver disease, diabetes mellitus
- glycerol-coated stopper used in vacutainer phlebotomy tubes
- glycerol containing IV medications
-mannitol infusion
Reference method for LDL cholesterol
Ultracentrifugation with beta quantification
Method for calcium measurement
Arsenazo dye reacts with calcium in an acid solution to form a blue-purple complex.
Measured at 660nm
Electroenzymatic reaction used in glucometer?
Glucose + O2 –(Glucose oxidase)–> Gluconic acid + H2O2
Current generated in the test strip is proportional to glucose conc
Light sources used in spectrophotometry
- Incandescent tungsten or tungsten-iodide lamp (visible and near IR)
- Deuterium (for UV)
- Mercury lamps (UV to mid visible)
Monochromators used in Spectrophotometry
- Coloured glass filters
- interference filters
- simple glass prism
- Diffraction gratings
Types of photodetectors used in spectrophotometry
- Photocell (no amplification)
- Phototube (outside voltage applied)
- Photomultiplier tube (series of anodes with increasing voltage - 200x more sensitive than phototube)
- Photodiode array (excellent linearity and speed)
Background correction is much more necessary for which type of AAS?
Electrothermal techniques
More than for flame AAS
What is the Zeeman correction
Correction of background absorbance for flameless AAS which utilises a magnetic field.
Magnetic fiels splits atomic energy levels into 2 components: polarised parallel and perpendicular
Absorption measurements at different polariser settings
Interferences of AAS
- Spectral - closely absorbing atomic species
- Non-spectral , non specific. Matrix (may affect nebulisation; viscosity, surface tension, density; sample flow rate)
- Non-spectral, specific. e.g. phosphate may interfere with calcium due to formation of calcium phosphate (overcome by adding cation e.g. strontium)
Advantages of AAS (Flame and Graphite)
- Low set up cost
- Simple sample preparation
- Few interferences
Disadvantages of AAS (Flame + graphie)
- Single element technique
- limited analytical range
Which form of AAS has a higher detection limit?
Flame AAS has higher detection limit than graphite furnace
i.e. less sensitive technique
How is chemiluminescence different to fluorescence
- no excitation radiation is required
- no monochromators are needed as CI arises from one species
Chemiluminescence reactions are oxidation reactions of …
- Luminol
- Acridium esters
- Dioxetanes
Advantages of Chemiluminescence
- subpicomolar detection limits
- speed
- ease of use
- simple instrumentation
Disadvantages of chemiluminescence
Impurities may cause a B/G signal –> reduce sensitivity, specificity
What is electroendosmosis
Movement of buffer ions and solvent relative to the fixed support
Ionisation technique used in GCMS
Electron ionisation - Electric potential applied which bombards molecules in source with high energy electrons
Ionisation techniques used in LC MS
- Electrospray
- Atmospheric pressure chemical ionisation
- MALDI
What does MALDI stand for
Matrix Assisted Laser Desorption Ionisation
When should a sweat test be delayed?
- Patient <2 weeks of age or <3kg
- Unwell
- dehydration or oedema
- corticosteroid treatment
How does neuraminidase affect ALP isoenzyme separation by electrophoresis?
Removes sialic acid residues.
Separation of bone and liver isoenzymes.
Bone is slowed down more than liver
What is the coloured molecule formed in the ALP enzymatic method?
p-nitrophenol
Yellow colour
The rate of absorbance increase of p-nitrophenol is measured at what wavelength?
404nm
Causes of increased osmolar gap
ethanol
methanol
ethylene glycol
polyethylene glycol (IV lorazepam)
propylene glycol (IV lorazepam, diazepam and phenytoin)
glycine (TURP syndrome)
maltose (IV IG – Intragram)
mannitol
sorbitol
Method for renin measurement
Sandwich immunoassay
Method for plasma renin activity involves measuring the amount of…
Angiotensin I
Generated after incubation of plasma at 37C, buffered to pH6 for optimal activity.
Endogenous angiotensiongen is utilised
Causes of decreased renin:
- Medications: beta blockers, methyldopa, clonidine, NSAIDs, OCP
- sodium loading
- renal impairment
- luteal phase
Conditions associated with a higher concentration of HbF
- Pregnancy
- Beta thalassaemia
- Sickle cell anaemia
- Leukaemia
- Insulin therapy
Non glucose adducts the Hb beta chain can occur via..
- Carbamylation (increased urea in RF)
- Acetylation (aspirin, alcohol, vit C)
Reference method for HbA1c
HPLC + MS
or HPLC + capillary electrophoresis
How does FTIR differ from simple IR spectroscopy
- it uses an interferometer - causes the IR radiation to interfere with itself.
- Computer software uses data from the interference of waves with different frequencies to determine the frequencies of IR that were absorbed by the sample.
What are the disadvantages of wet chemical analysis for renal calculi analysis?
- requires at least 10-15mg of sample
- only identifies ions present not the specific compounds
- can miss rare material
What is the main disadvantage of using Nitroprusside to measure ketones?
Beta-hydroxybutarate does not react with nitroprusside
Nitroprusside is most sensitive at measuring which type of ketone
Acetoacetate
Nitroprusside + Acetoacetate (or acetone) -> Red colouration
The enzymatic method for ketone analysis uses which enzyme
Beta hydroxybutarate dehydrogenase
Beta HB + NAD –> Acetoacetate + NADH. Change in absorbance at 340nm
Point of care device for Beta hydroxybutarate uses …
Electrochemical sensor.
Enzyme: Beta HB dehydrogenase
Generated NADH reoxidised to NAD- and current is measured
Glucose oxidase method for glucose:
Glucose + O2 –>
Gluconic acid + H2O2
Hexokinase method for glucose:
1) Glucose + ATP –> __________ + ADP
2) _________________ + NADP –> 6-phosphogluconate + NADPH + H+
Glucose- 6 Phosphate
Point of care device using glucose oxidase involves measurement of what coloured chromogen
Oxidised ortho-dianisidine
Formed by reaction with H2O2
Disadvantages of POC glucose
- No reference method for whole blood glucose
- Affected by medications, oxygen therapy, anaemia, haematocrit
- Not easily connected to LIS
- no Medicare rebate
- errors due to lack of expertised and insufficient QC
What does ELISA stand for
Enzyme linked immunosorbent assay
Method for chromagranin A
ELISA
Sandwich immunoassay incorporating 2 monoclonal Abs targeting different epitopes
Newborn screening for CF uses
Immunoreactive trypsinogen
In the enzymatic method of ammonia, what is the enzyme used?
Glutamate dehyrodgenase (GLDH)
Change in absorbance measured at 340nm
Why does plasma ammonia concentration increase after collection?
Deamination of plasma proteins and amino acids by enzymes
Increases with higher GGT concentrations
Interferences in enzymatic assay for Lactate
- Haemolysis
- Elevated bilirubin
- Glycolic acid
- N-acetyl cysteine
What is the effect of excess heparin in the blood gas tube when measuring HCO3?
Decreases the pCO2 and calculated HCO3-
Method for bone ALP
2 site immunoassay
Reference method for ALP
IFCC kinetic spectrophotometry
Substrate = 4-nitrophenyl phosphate
Types of tissue non-specific ALP
- Bone
- Liver
- Kidney
Buffer used in IFCC reaction for ALP measurement
AMP (2- amino-2-methyl-1-propanol)
Transphosphorylating buffer (accepts PO4) and increases reaction rate
In measuring ALP isoenzymes, addition of neuraminidase separates which forms?
Bone and liver
How does neuraminidase separate bone and liver isoenzymes?
Removes sialic acid. Bone contains more sialic acid than liver –> greater reduction in mobility
Reference method for glucose
Isotope dilution LC-MS
Albumin is measured at what wavelength?
604nm
Reference method for albumin
IFCC Optimized immunoturbidimetry/nephelometry
Reference method for total protein
INSTAND spectrophotometric method based on biuret reaction
Interferences in biuret method for total protein
- Haemolysis, Icterus, Lipaemia
- Dextran
- Contamination with ammonium ion
Reference method for ALT and AST
IFCC kinetic spectrophotometry with P5P
Method used for Cystatin C
Immunonephelometry or immunoturbidimetry
What is the dye formed in the Trinder reaction
quinoneimine dye
Reference method for Total cholesterol
Isotope Dilution MS
Reference method for Triglycerides
Isotope Dilution MS
U/L of enzyme = ________ of substrate consumed per minute
micromol of substrate
Formula for correcting calcium for albumin
Corrected calcium = Measured calcium – ((Albumin-41) x 0.02)
Reference method for total calcium
ICP-MS
When does total calcium not reflect free/ionised calcium?
- abnormal protein concentration
- when patient has acid-based disturbance (higher H+ -> higher free Ca2+)
The Payne formula for Calcium correction was derived using which method for albumin measurement
BCG
If a lab uses BCP, this can affect the performance of BCG-equations
Enzyme that conjugates bilirubin
UDP Glucuronosyltransferase 1A1 (UGT1A1)
What does NIST stand for
National Institute of Standards and Technology
What is a diazo group
An organic moiety consisting of 2 linked Nitrogen atoms (azo) at the terminal position
Interferences in Diazo method
- Haemoglobin
- Lipaemia
- Paraproteins
- Ascorbic acid
- Indican
- Indocyanine green
Reaction for enzymatic measurement of bilirubin
Vanadate OR Bilirubin Oxidase - oxidises
Bilrubin –> Biliverdin
Monitor decrease in absorbance
Most common method for Lithium measurement
Colourimetric
(Abbott - substituted porphyrin reacts with Lithium)
Alkaline pH required
Reference method for Lithium
ID-ICP-MS
What is heparin
A mucoitin polysulfuric acid
Accelarates action of antithrombin III
What is Daratumumab?
Human monoclonal IgG1 anti-CD38
Used in treatment of multiple myeloma
Reasons for derivitisation
- improve performance in chromatographic environment
- thermal stability
- volatility
- improve separation
- improve sensitivity of detection
Identification of CSF by beta trace protein can be limited in what situations
- Bloody discharge - low levels may be found in blood
- Meningitis - may reduce beta trace protein levels
Analytical issues with serum free light chains - name 5
- Interference from turbidity/lipaemia
- Hook effect
- Non-linearity
- Reagent lot to lot variation
- Results can differ between methods (polyclonal vs monoclonal Abs)
In CKD, different K/L ratio RI may be needed depending on assay
Method for plasma free metanephrines
LCMS-MS
Impact of OCP on AMH levels
Decrease by approx 10%
In a chemiluminescence reaction (acridium ester),
Dye system + H202 –> ??
2H20 + oxidised dye + LIGHT
Light is emitted when the electron returns to the ground state
Assays (Abbott) affected by High Bilirubin
- Cholesterol - decrease
- Trigs - increased
- Amylase - increased
Medications that increase 5-HIAA measurements
- Paracetamol
- Phenacetin
- Glyceryl guaiacolate (found in many cough syrups)
Properties of a good internal standard
- similar mass of the analyte of interest
- similar ionisation potential
- not present in clinical samples
- not affected by spectroscopic interferences
- not a source of analyte contamination
Common internal standards used in ICP-MS
- Scandium
- Germanium
- Rhodium
- Yttrium
Anti-hypertensive that do not affect ARR
- verapamil
- prazosin
- hydralazine
- moxonidine
Definition of Km (Michaelis-Menten constant)
Concentration at half the maximum velocity of the enzymatic reaction
Difference between enzyme activators and coenzymes
- Enzyme activators: usually ions that increase the activity by facilitating more stable form of enzyme e.g. Mg++ for CK
- Coenzymes usually more complex that activators but smaller than enzymes themselves e.g. NAD, NADP, P-5-P
How can a Propofol infusion affect lipid results?
Propofol infusions (used in ICU as sedative) contain glycerol
Can result in falsely high triglyceride results
Types of detectors used for measuring radioactivity
- Gas filled
- Scintillation - more common
- absorption of radiation produces a flash of light
- liquid or crystal
Enzymatic method of HbA1c uses which enzyme
FPOX (Fructosyl peptide oxidase)
What formula is used (by Sonic) to calculate free testosterone?
Vermuelen formula
accounts for both SHBG and albumin
Name 4 equations for calculating free testosterone and the components they include
- Vermuelen - T, SHBG, albumin
- Sodergard - T, SHBG, albumin
- Nanjee-Wheeler - T, SHBG
- Ly-Handelsman - T, SHBG
What does PETIA stand for?
Particle Enhanced Turbidimetric Immunoassay
What does PETINIA stand for?
Particle Enhanced Turbidimetric Inhibition Immunoassay
What does CEDIA stand for
Cloned Enzyme Donor Immunoassays
What does ELISA stand for
Enzyme Linked Immunosorbent Assays
Cause of falsely low phosphate result
IV Mannitol (e.g. for high ICP)
Interferes with reaction of phosphate with molybdate
Preferred specimen for LD?
Serum
Plasma has higher LD; released from platelets when they activate
Conditions of standard gel electrophoresis
- Agarose gel medium (typically 0.5-2% concentration); low endosmosis effect
- buffer pH 8.6 (constant) - most proteins negative charged -> anode
What does EDTA stand for?
Ethylene-diamine-tetraacetic acid
Cause of clear green appearing serum with positive lipaemic index
Patent blue dye
Used in sentinal lymph node biopsy
Types of analysers available for Mass Spec
- Quadrupole
- Ion trap
- Tandem mass spec
- TOF
- Orbitrap
TOP and Orbitrap are types of “high resolution MS”
Most common type of detector used in MS
Electron multiplier
Ionisation types used in LC/MS
- Electrospray
- Atmospheric pressure chemical ionisation (APCI)
- MALDI
Types of CK isoenzymes
- CK-BB - brain, lung, intestine
- CK-MB - cardiac muscle
- CK-MM - skeletal muscle
Above located in cytosol
CK-mitochondria - can occur w necrosis
Types of Macro CK
- MacroCK 1 - typically CK-BB with IgG. Females >50 years
- MacroCK 2 - complexes of mitochondrial CK
What is derivitisation and why is it used?
Chemical modification of the analyte (e.g. alkylation, Silylation).
Used to improve:
- volatility and thermal stability in gas chromatography
- retention and resolution from interferences
- detection (e.g fluorescence detection)
Formula for theoretical plates
N = 16 (t / w) ^2
where t is retention time
and w is width of peak
Formula for Height of theoretical plates (HETP) in chromatography
HETP = column length / N
where N is number of theoretical plates
How to increase number of theoretical plates in chromatography
- longer column
- smaller average packing particle size
- optimising flow rates
- higher mobile phase viscosity
- smaller sample volume
Buffer used in SPIFE method for serum protein electrophoresis
Tris buffer
Indicator dye used on urine dipstick for protein detection
Bromphenol blue
Difference between isoenzyme and isoform
Both catalyse the same reaction
* Isoenzyme - different genetic loci
* Isoform - same genetic loci but different post translational modifications
