Analytical Flashcards
What is the Friedewald Equation?
LDL-Cholesterol= Total Cholesterol- HDL-cholesterol - TG/2.2
Assumptions
- Chylomicrons absent (fasting)
This calculation is invalid when TG>= 4.5 mmol/L
When TG is >= 4.5 mmol/L <14.6 mmol/L LDL is measured
Name substances which interfere with Total Cholesterol enzymatic method. What is the equation?
Cholesteryl ester + H20»_space; CE hydrolase» Cholestreol + FA
Cholesterol + O2» Chol Oxidase» Cholest-4-en-3-one + H202
H202 + TODB + 4 Aminoantipyrine»_space;perxidase» Quinoneimine dye
A @604
Interference
Plant sterols
Bilirubin, Vit C & haemoglobin compete with the oxidation reaction.
High biological variation
What is the HDL reaction equation? Name interference’s
Phase 1 Accelerating the non-HDL unesterified cholesterol
Phase 2
HDL cholesterol Plus HDL detergent to disrupt HDL
HDL Cholesterol + H20 + O2»_space; CHE and CHO Cholest-4-en-3-one + H2O2
H2O2 + TODB + 4 -AAP»_space; Peroxidase» Quinoneimine dye + 2H2O
Interference
TG > 22.6 mmom/L
Immunoglobulins - may falsley increase result
Vit C
Liver Cirrhosis, dyslipidaemia – Can measure via an alternate method.
What is the Triglyceride reaction equation? Name interferences.
TG + H2O»_space; Lipoprotein lipase» Glycerol + FFA
Glycerol + ATP»_space; Glycerol Kinase + Mg» Glycerol-3-phosphate + ADP
Glycerol-3-phosphate + O2»_space; GPO» Dihyroxyacetone phosphate + H2O2
H2O2 + 4-aminoantipyrine + 4- Chorophenol»_space; PO» Quinoneimine dye
A @ 500nm
NB/ No glycerol blank- TG is slightly overestimated for endogenous glycerol.
Interferences
Bilirubin - Neg
N-Acetyle-L-Cysteine (NAC)
Haemolysis
Endogenous glycerol - elevated in Hyperthyroidism , CRF, DM, Liver disorders
What is the urea reaction equation? Name interferences.
Urea + H2O»_space; Urease» @NH4+ + CO2
a-Ketoglutarate + NH4+ + NADH»_space; GLDH» L-Glutamet + NAD+ + H2O
Decrease in A@340nm
Interference
Urine- Endogenous Ammonium ions. Elevated levels may be seen in acidosis.
Ammonia- Air exposure, Age of urine sample
What is the Urea ref method?
Isotope depletion MS
Name 4 HDL methods
Ref Method - CDC- two step ultracentifugation method followed by cholesterol analysis.
Centrifugation for up to 18 hours @ 10’C, 40 000rpm
Immunoseparation- Anti-human lipoprotein antibody binds and inactivates each of the other fractions. Cholesterol esterase and oxidase then react with HDL.
Polyethylene glycol-modified enzyme/a-cyclodextrin sulfate (PEGME)- sulphated cyclodextrin and dextran sulphate form water-soluble complexes with LDL, VLDL and chylomcirons making them resiatnt to PEG modified enzymes, in the 2nd reaction step HDL-Chol levels are mearsured byt the actionof chol esterase and oxidase.
Clearance method - Selective ionic strength buffer releases chylomicrons, LDL and VLDL components and then removes them by the action of cholesterol esterase and oxidase. HDL-cho, is then released in the 2nd step of the reaction by surfactants and then removed by enzymes.
What is the LDL - reference method?
B-quantification electrophoresis
What is the reaction equation for HCO3?
PEP + HCO3-»_space;> PEPC» Oxaloacetate + H2PO4-
Oxaloacetate + NADH analog + H+»_space; MDH» Malate + NAD+ analog.
Decrease in A @ 340nm
How is Bicarb calculated in POCT?
Calculated HCO3- = 0.23 X pCO2 X 10^(pH-pKp)
Where pKp = 6.095
What is the Reaction equation for Total Protein?
Biuret reaction
Divalent copper (Cu2+) + peptide bonds»_space; Purple coloured complex
A@572nm
Venous stasis during collection will artefactually increase total protein.
Supine collection will lower TP levels due to fluid distribution.
Plasma level 3-5g/L higher than serum do to fibrinogen.
What is the urine protein reaction equation? Name interferences
Urine protein + benzethonium chloride»_space;ALk pH» protein denaturation
Increase in turbidity»_space;> A@404nm
Interferences
Low - urine collected with acid preservative
High- Fluorescein (CSF/Urine)
- Gelatin based plasma expanders e.g haemocell, gelofusine
24hr protein = urine protein g/L X 24hr urine volume (L) = g/day
What is the reaction equation for GGT? Name intereferences
L-g-Glutamyl-3-carboxy-4-nitronanilide + glyclglycine»_space; GGT»> 3-carboxy-4-nitroaniline + L-g-glutamyl-glycylglycine
change in A@416nm
Interference
Macro-GGT
IgM paraprotein causing Ig-GGT formation complexes
What is the reaction equation for ALP?
p-Nitropheyl phosphate + H20»_space; ALP,Mg2+,Zn2+» Phosphate = pNitropenol (benzenoid form)»ALK pH»> Yellow quionoid form
A@404nm
Interference
EDTA
Oxalate
Haemolysis
What is the reaction equation for conjugated bilirubin? Name interferences
Conjugated bilirubin + diazotized suphanic acid»_space;> azobilirubin complex —Direct method
A@546nm
Interferences
haemolysis, Lipaemia, Indocyanine green diagnostic imaging dye, draw sample >2hrs administration
What is the ref method for Bilirubin?
based on Jendrassik-Grof method
What is the reaction equation for CK? Why is NAC used ?
Why is the [Mg] important?
Creatine phosphate + ADP»_space;>CK, Mg» ATP + creatine
Glucose + ATP»_space;>hexokinase» glucose-6-phosphate + ADP
G-6-P + NAD»_space; G6PD» 6-phosphogluconolactone + NADH
A@340nm
CK in plasma in relatively unstable - NAC partially restores CK activity.
Mg is important co-factor in the reaction - optimal Mg range is narrow, excess is inhibitory
Haemolysis - can cause xreactivity with adenylate kinase in RC.
What are the CK-isoenzymes and how do we distinguish between them?
CKMM(Sket muscle), CKMB(cardiac muscle), CKBB(brain and intestine)
Electrophoresis as isoenzymes have different electrophoretic mobilities, forming separate bands in the gel.
What is the LDH reaction equation? Name interference’s
L-Lactate + NAD»>LDH»> pyruvate + NADH
A@340nm
Interferences
Haemolysis - ~150 higher in RBC
EDTA
Cold temp (LD4 and LD5 are cold labile - false low results)
Platelet contamination - incomplete centrifugation
Macro-LDH
Describe beers law?
A = ExBxC. E= molar absorptivity , b= path length c=concentration of absorbing molecules
When light passes through or is reflected by a sample, the amount of light absorbed is the difference between the incident light and transmitted light.
Name 3 types of light sources in spectrophotometry
Deuterium
Tungsten-halogen
Xenon Flash lamp
How is Ion exchange HPLC used for glycated Hb?
uses buffers of increasing ionic strength to seperate HBA1c from other Hb species by difference in charge. The concentration of each fraction is quantified by calculating the area under the peak
-ability to detect most common Hb variants
- can have some variants co-elute, labour intensive long TAT
Describe the enzymatic method from HBA1c
Uses enzyme cleaving the N-terminal valine.
Lysed RBC undergo proteolytic digestion releasing the substrate for frutosyl-valine oxidase which produces H2O2. measured using horseradish peroxidase to produced chromogen (trinder)
Unaffected by variants, automatable, POCT, low TAT
No presumptive ID for variants, other trinder reaction interferences
Describe the IA for HBA1c
use Ab which target the 4-10a.a on the valine terminal end of the b-chain. Can be immunoturbidometric, latex -enhanced competitive immunoturbidometric.
Automatable, low TAT, no additional labour cost, can be affected by variants if the mutation occurs in this region, no presumption variant ID
Describe boronate affinity HPLC?
separation based on structural difference.
m-aminophenylboronic acid binds all glycated Hb to affinity resin. Is considered to have the least interences from Hb variants.
-if Hb variants are glycated will also bind. No presumpitive ID
Describe capillary electrophoresis for HBA1c
Separated by charge and EOF. Charged molecules are highly resolved by their electrophoretic mobility and their separation depends on electrolyte pH and electroosmotic flow - CE
Can Id Hb variants and does not have analytical interference from common variants, automatable - continous leading to fast TAT, Throughput a disadvantage.
Describe serum protein electrophoresis
A method for separating proteins based their physical properties. Serum protein is placed on a specific medium, and a charge is applied. The net charge and the size and shape of the protein separates the proteins present into bands.
Albumin is the most negatively charged protein and the most abundant travels closer to the Anode (POS), followed by
Alpha1 - alpha1-antitrypsin, thyroid-binding globulin and transcortin.
Alpha2- alpha2 macroglobulin, haptoglobin, ceuloplasmin
Beta1 - transferrin
Beta2 - lipoprotein
Gamma - IgA, IgG, IgM
Multiple myeloma is characterized by a narrow spike in the gamma, beta, or aplha2 regions, usually greater than 3g per dL and BM with 10-15% plasma cell involvement, and skeletal lesions.
Anaemia, pancytopenia, hypercalcemia and renal disease.
Why can an indirect ISE Na+ be affected by lipid and protein concentrations?
Indirect ISE assumes a plasma water of 93%. Sample is diluted with ICT fluid prior to measurement. This dilution is amplified when lipids >2g/L, and protein <40 or >100g/L
What are theoretical plates?
Theoretical plates are instantaneous unmeasurable equilibrium points within a column. The number of theoretical plates can however be calculated, and used to measure the efficiency of how well the column separates a mixture of compounds.
N=16(tr/W)^2
The larger the number of theoretical plates the better the separation of the compounds- therefore the narrower the peaks, better the resolution and better ID
Discuss the principle of HPLC
Method used to separate molecules from complex mixtures for either analytical or prepartive purposes. In HPLC the stationary phase is in a cololumn packed with silica beads, the mobile phase liquid is then passed through the packed stationary phase to achieve separation. HPLC uses an automated pump at high pressure ~5000psi.
Separation can be based on Size exclusion, hydrophobicity(Partition), Ion exchange
What is endosmosis?
Movement of solvent and its solutes relative to fixed support is referred to as endosmosis»_space;Preferential movement of water in one direction.
